Canine IL-13 immunoregulatory proteins and uses thereof

ABSTRACT

The present invention relates to canine interleukin-4, canine or feline Flt-3 ligand, canine or feline CD40, canine or feline CD154, canine interleukin-5, canine interleukin-13, feline interferon alpha, and/or feline GM-CSF proteins; to canine interleukin-4, canine or feline Flt-3 ligand, canine or feline CD40, canine or feline CD154, canine interleukin-5, canine interleukin-13, feline interferon alpha, and/or feline GM-CSF nucleic acid molecules, including those that encode canine interleukin-4, canine or feline Flt-3 ligand, canine or feline CD40, canine or feline CD154, canine interleukin-5, canine interleukin-13, feline interferon alpha, and/or feline GM-CSF proteins, respectively; to antibodies raised against such proteins; and to inhibitory compounds that regulate such proteins. The present invention also includes methods to identify and obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies and/or inhibitory compounds as well as the use of such therapeutic compositions to regulate an immune response in an animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/322,409, filed May 28, 1999, entitled “CANINE AND FELINEIMMUNOREGULATORY PROTEINS, NUCLEIC ACID MOLECULES, AND USES THEREOF”;which claims priority to U.S. Provisional Patent Application Ser. No.60/087,306, filed May 29, 1998, entitled “CANINE INTERLEUKIN-4 AND FLT-3LIGAND PROTEINS, NUCLEIC ACID MOLECULES, AND USES THEREOF”; each ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to canine interleukin-4, canine or felineFlt-3 ligand, canine and feline CD40, canine or feline CD154, canineinterleukin-5, canine interleukin-13, feline interferon alpha, or felineGM-CSF nucleic acid molecules, proteins encoded by such nucleic acidmolecules, antibodies raised against such proteins and/or inhibitors ofsuch proteins or nucleic acid molecules. The present invention alsoincludes therapeutic compositions comprising such nucleic acidmolecules, proteins, antibodies and/or inhibitors, as well as their useto regulate an immune response in an animal.

BACKGROUND OF THE INVENTION

Regulating immune responses in animals is important in diseasemanagement. Immune responses can be regulated by modifying the activityof immunoregulatory molecules and immune cells.

Several immunoregulatory molecules have been found in humans and othermammal species. Interleukin-4, produced by activated type 2 helper cells(T_(H)2 cells), has a number of functions. These functions includepromotion of naive T cells and B cells to differentiate and proliferate.IL-4 promotes T_(H)2 differentiation and inhibits T_(H)1 development.FMS-like tyrosine kinase 3, (Flt-3 ligand) stimulates the expansion andmobilization of hematopoetic precursor cell stimulating activity. CD40is a type I transmembrane protein expressed on antigen presenting cells,such as B lymphocytes, and other types of cells such as endothelialcells, epithelial cells, and fibroblasts. CD40 ligand (also known asCD154) is a type II transmembrane protein that is preferentiallyexpressed on activated T lymphocytes. The CD40-CD154 interactionregulates diverse pathways of the immune system, including B cellproliferation, immunoglobulin production and class switching by B cells,activation and clonal expansion of T cells, activity of antigenpresenting cells, growth and differentiation of epithelial cells, andregulation of inflammatory responses at mucosal and cutaneous sites.Interleukin-5 is produced by activated type 2 helper cells (T_(H)2),mast cells, and eosinophils. Its main functions include promotion ofgrowth and differentiation of eosinophils and generation of cytotoxic Tcells from thymocytes. Interleukin-13 is produced by T_(H)1 and T_(H)2cells, and promotes growth and differentiations of B cells,up-regulation of MHC class II and CD23 expression onmonocytes/macrophages and B cells; and inhibition of production ofinflammatory cytokines such as IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12,among others. Interferon alpha is an antiviral protein that has threemajor functions: it inhibits viral replication by activating cellulargenes that destroy mRNA and inhibit protein translation, it induces MHCclass I expression in non virally-infected cells, increasing resistanceto NK cells, and can activate NK cells. GM-CSF, (granulocyte-macrophagecolony-stimulating factor) stimulates the production of granulocytes andmacrophages.

Prior investigators have disclosed sequences encoding feline IL-4(Lerner et al., Genbank Accession No. U39634); porcine L-4 (Zhou et al.,Genbank Accession No. L12991); bovine IL-4 (Heussler, V.T., et al.,Gene. vol. 114, pp. 273-278, 1992); ovine IL-4 (Seow, H.-F., et al.,Gene, vol. 124, pp. 291-293, 1993); human IL-4 (Yokota, T., et al.,Proc. Natl. Acad. Sci. U.S.A., vol. 83(16), pp. 5894-5898, 1986); andmurine IL-4 (Sideras, P., et al., Adv. Exp. Med. Biol., vol. 213, pp.227-236, 1987). Prior investigators have disclosed sequences encodingmurine Flt-3 ligand (McClanahan et al., Genbank Accession No. U44024);and human Flt-3 ligand (Lyman et al., Blood, vol. 83, pp. 2795-2801,1994). Prior investigators have disclosed sequences encoding human CD40(Stamenkovic et al., EMBO J., vol. 8:1403-1410, 1989, GenBank AccessionNo. (X60592), bovine CD40 (Hirano et al., Immunology, vol. 90, pp.294-300, 1997), GenBank Accession No. U57745), and murine CD40 (Grimaldiet al., J. Immunol., vol. 143, pp.3921-3926, 1992; Torres and Clark, J.Immunol., vol. 148, pp. 620-626, 1992, GenBank Accession No. M83312).Prior investigators have disclosed sequences encoding human CD154 (Grafet al., Eur. J. Immunol., vol. 22, pp. 3191-3194, 1992; Hollenbaugh, etal., EMBO J., vol. 11:4313-4321, 1992; Gauchat et al., FEBS lett., vol.,315, pp. 259-266, 1993; GenBank Accession Nos L07414, X68550, Z15017,X67878, respectively); bovine CD154 (Mertens et al., Immunogenetics,vol. 42, pp. 430-431, GenBank Accession No. Z48468); and murine CD154(Armitage et al., Nature, vol. 357, pp. 80-82; 1992, GenBank AccessionNo. X65453). Prior investigators have disclosed sequences encodingfeline interleukin-5 (Padrid et al., Am. J. Vet. Res., vol. 59, pp.1263-1269, 1998, GenBank Accession No. AF025436) and human interleukin-5(Azuma et al., Nucleic Acids Res., vol. 14, pp. 9149-9158, 1986, GenBankAccession No. X04688). Prior investigators have disclosed sequencesencoding human interleukin-13 (McKenzie et al., Proc. Natl Acad. Sci.USA, vol. 90, pp. 3735-3739, 1993; Minty et al., Nature, vol. 362, pp.248-250, 1993, GenBank Accession Nos L06801 and X69079, respectively);murine interleukin-13 (Brown et al., J. Immunol., vol. 142, pp. 679-687,1989, GenBank Accession No M23504); and rat interleukin-13 (Lakkis etal., Biochem. Biophys. Res. Commun., Vol. 197, pp. 612-618, 1993,GenBank Accession No. L26913). Prior investigators have disclosedsequences encoding feline interferon (Nakamura, N., Sudo, T., Matsuda,S., Yanai, A., Biosci. Biotechnol. Biochem. (1992)Vol: 56 pp 211-214,GenBank accession # E02521). Prior investigators have also disclosedsequences encoding feline GM-CSF (direct submission to GenBank,Accession No. AF053007)

There remains a need for compounds and methods to regulate an immuneresponse by manipulation of the function of canine interleukin-4, canineor feline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF.

SUMMARY OF THE INVENTION

The present invention relates to canine interleukin-4, canine or felineFlt-3 ligand, canine or feline CD40, canine or feline CD154, canineinterleukin-5, canine interleukin-13, feline interferon alpha, or felineGM-CSF nucleic acid molecules, proteins encoded by such nucleic acidmolecules, antibodies raised against such proteins and/or inhibitors ofsuch proteins or nucleic acid molecules. Identification of the nucleicacid molecules of the present invention is unexpected because initialattempts to obtain nucleic acid molecules using PCR were unsuccessful.After numerous attempts, the inventors discovered specific primers thatwere useful for isolating such nucleic acid molecules.

One embodiment of the present invention is an isolated nucleic acidmolecule selected from the group consisting of: (a) an isolated nucleicacid molecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:19, and/or SEQ ID NO:21 or a homolog thereof, wherein said homologhas an at least about 50 contiguous nucleotide region identical insequence to a 50 contiguous nucleotide region of a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:19, and/or SEQ ID NO:21; (b) an isolatednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:36, and/or SEQ ID NO:37 or a homolog thereof, whereinsaid homolog has an at least 40 contiguous nucleotide region identicalin sequence to a 40 contiguous nucleotide region of a nucleic acidmolecule having a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:36, and/or SEQ ID NO:37; (c) an isolated nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, and/or SEQ ID NO:50, and/or a homologthereof, wherein said homolog has an at least 30 contiguous nucleotideregion identical in sequence to a 30 contiguous nucleotide region of anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and/or SEQ ID NO:50;(d) an isolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and/or SEQ ID NO:59,and/or a homolog thereof, wherein said homolog has an at least 40contiguous nucleotide region identical in sequence to a 40 contiguousnucleotide region of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, and/or SEQ ID NO:59; (e) an isolated nucleicacid molecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:60 and/or SEQ ID NO:62, and/or a homologthereof, wherein said homolog has an at least 30 contiguous nucleotideregion identical in sequence to a 30 contiguous nucleotide region of anucleic acid molecule having a 3 nucleic acid sequence selected from thegroup consisting of SEQ ID NO:60 and/or SEQ ID NO:62; (f) an isolatednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69 and/or SEQ ID NO:71, and/or a homologthereof, wherein said homolog has an at least 45 contiguous nucleotideregion identical in sequence to a 45 nucleotide region of a nucleic acidmolecule having a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67,SEQ ID NO:68, SEQ ID NO:69 and/or SEQ ID NO:71; (g) an isolated nucleicacid molecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, and/or SEQ ID NO:79, and/or a homolog thereof, whereinsaid homolog has an at least 35 contiguous nucleotide region identicalin sequence to a 35 contiguous nucleotide region of a nucleic acidmolecule having a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, and/or SEQ ID NO:79; (h) an isolated nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85,and/or SEQ ID NO:87, and/or a homolog thereof, wherein said homolog hasan at least 45 contiguous nucleotide region identical in sequence to a45 contiguous nucleotide region of a nucleic acid molecule having anucleic acid sequence selected from the group consisting of SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and/orSEQ ID NO:87; (i) an isolated nucleic acid molecule comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:102, SEQ ID NO:103, SEQ ID NO:104, and/or SEQ ID NO:106, and/or ahomolog thereof, wherein said homolog has an at least 15 contiguousnucleotide region identical to a 15 contiguous nucleotide region of anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ IDNO:104, and/or SEQ ID NO:106; (j) an isolated nucleic acid moleculehaving a nucleic acid sequence selected from the group consisting of SEQID NO:107, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113,SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:155, SEQ IDNO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:163, SEQID NO:164, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:170,and SEQ ID NO:172; and/or (k) an isolated nucleic acid molecule having anucleic acid sequence selected from the group consisting of SEQ IDNO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQID NO:126.

Another embodiment of the present invention is an isolated nucleic acidmolecule selected from the group consisting of: (a) a nucleic acidmolecule having a nucleic acid sequence that is at least about 92percent identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:19, and/or SEQ ID NO:21; (b) a nucleic acid molecule having a nucleicacid sequence that is at least about 75 percent identical to a nucleicacid sequence selected from the group consisting of SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:36, and/or SEQ ID NO:37;(c) a nucleic acid molecule having a nucleic acid sequence that is atleast about 75 percent identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and/or SEQ IDNO:50; (d) a nucleic acid molecule having a nucleic acid sequences thatis at least about 70 percent identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and/or SEQ ID NO:59;(e) a nucleic acid molecule having a nucleic acid sequence that is atleast about 70 percent identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO:60 and/or SEQ ID NO:62; (f) anucleic acid molecule having a nucleic acid sequence that is at leastabout 85 percent identical to a nucleic acid sequences selected from thegroup consisting of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, and/or SEQ ID NO:71; (g) a nucleicacid molecule having a nucleic acid sequence that is at least about 91percent identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,SEQ ID NO:77, and/or SEQ ID NO:79; (h) a nucleic acid molecule having anucleic acid sequence that is at least about 90 percent identical to anucleic acid sequence selected from the group consisting of SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and/orSEQ ID NO:87; (i) a nucleic acid molecule having a nucleic acid sequencethat is at least about 65 percent identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:102, SEQ IDNO:103, SEQ ID NO:104, and/or SEQ ID NO:106; (j) a nucleic acid moleculehaving a nucleic acid sequence that is selected from the groupconsisting of SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118, SEQID NO:155, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:161,SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:167, SEQ IDNO:169, SEQ ID NO:170 and/or SEQ ID NO:172; and/or (k) a nucleic acidmolecule having a nucleic acid sequence that is selected from the groupconsisting of SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ IDNO:123, SEQ ID NO:124, and/or SEQ ID NO:126.

Yet another embodiment of the present invention is an isolated nucleicacid molecule selected from the group consisting of: (a) a nucleic acidmolecule having a nucleic acid sequence encoding an IL-4 proteinselected from the group consisting of (i) a protein having an amino acidsequence that is at least about 85 percent identical to an amino acidsequence selected from the group consisting of SEQ ID NO:2 and/or SEQ IDNO:20 and/or (ii) a protein comprising a fragment of at least 20 aminoacids of an amino acid sequences selected from the group consisting ofSEQ ID NO:2 and/or SEQ ID NO:20; (b) a nucleic acid molecule having anucleic acid sequence encoding a Flt-3 ligand protein selected from thegroup consisting of (i) a protein having an amino acid sequence that isat least about 75 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26,SEQ ID NO:31, and/or SEQ ID NO:34 and/or (ii) a protein comprising afragment of at least 25 amino acids of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26,SEQ ID NO:3 1, and/or SEQ ID NO:34; (c) a nucleic acid molecule having anucleic acid sequence encoding a Flt-3 ligand protein selected from thegroup consisting of (i) a protein having an amino acid sequence that isat least about 75 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:44 and/or SEQ ID NO:49 and/or(ii) a protein comprising a fragment of at least 25 amino acids of anamino acid sequence selected from the group consisting of SEQ ID NO:44and/or SEQ ID NO:49; (d) a nucleic acid molecule having a nucleic acidsequence encoding a CD40 protein selected from the group consisting of(i) a protein having an amino acid sequence that is at least about 70percent identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:53 and/or SEQ ID NO:58 and/or (ii) a proteincomprising a fragment of at least 30 amino acids of an amino acidsequence selected from the group consisting of SEQ ID NO:53 and/or SEQID NO:58; (e) a nucleic acid molecule having a nucleic acid sequenceencoding a CD40 protein selected from the group consisting of (i) aprotein having an amino acid sequence that is at least about 60 percentidentical to an amino acid sequence comprising SEQ ID NO:61 and/or (ii)a protein comprising a fragment of at least 20 amino acids of an aminoacid sequence comprising SEQ ID NO:61; (f) a nucleic acid moleculehaving a nucleic acid sequence encoding a CD154 protein selected fromthe group consisting of (i) a protein having an amino acid sequence thatis at least about 80 percent identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:65 and/or SEQ ID NO:70,and/or (ii) a protein comprising a fragment of at least 35 amino acidsof an amino acid sequence selected from the group consisting of SEQ IDNO:65 and/or SEQ ID NO:70; (g) a nucleic acid molecule having a nucleicacid sequence encoding a CD154 protein selected from the groupconsisting of (i) a protein having an amino acid sequence that is atleast about 85 percent identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:73 and/or SEQ ID NO:78, and/or (ii) aprotein comprising a fragment of at least 50 amino acids of an aminoacid sequence selected from the group consisting of SEQ ID NO:73 and/orSEQ ID NO:78; (h) a nucleic acid molecule having a nucleic acid sequenceencoding an IL-5 protein selected from the group consisting of (i) aprotein having an amino acid sequence that is at least about 85 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:81 and/or SEQ ID NO:86 and/or (ii) a protein comprising afragment of at least 20 amino acids of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:81 and/or SEQ ID NO:86; (i) anucleic acid molecule having a nucleic acid sequence encoding an IL-13protein selected from the group consisting of (i) a protein having anamino acid sequence that is at least about 70 percent identical to anamino acid sequence selected from the group consisting of SEQ ID NO:92,SEQ ID NO:97, SEQ ID NO:100, and/or SEQ ID NO:105 and/or (ii) a proteincomprising a fragment of at least 15 amino acids of an amino acidsequence selected from the group consisting of SEQ ID NO:92, SEQ IDNO:97, SEQ ID NO:100, and/or SEQ ID NO:105; (j) a nucleic acid moleculehaving a nucleic acid sequence encoding an interferon alpha proteinhaving an amino acid sequence that is selected from the group consistingof amino acid sequence SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQID NO:117, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165,SEQ ID NO:168, and/or SEQ ID NO:171; (k) a nucleic acid molecule havinga nucleic acid sequence encoding a GMCSF protein having an amino acidsequence that is selected from the group consisting of amino acidsequence SEQ ID NO:120, SEQ ID NO:125, and/or (1) a nucleic acidmolecule comprising a complement of any of said nucleic acid moleculesas set forth in (a), (b), (c), (d), (e), (f), (g), (h), (i), 0), and/or(k), wherein said IL-4 protein elicits an immune response against anIL-4 protein selected from the group consisting of SEQ ID NO:2 and/orSEQ ID NO:20 and/or is a protein with interleukin-4 activity, said Flt-3ligand protein elicits an immune response against a Flt-3 ligand proteinselected from the group consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ IDNO:26, SEQ ID NO:31, SE ID NO:34, SEQ ID NO:44, and/or SEQ ID NO:49and/or is a protein with Flt-3 ligand activity, said CD40 proteinelicits an immune response against a CD40 protein selected from thegroup consisting of SEQ ID NO:53, SEQ ID NO:58, and/or SEQ ID NO:61and/or is a protein with CD40 activity, said CD154 protein elicits animmune response against a CD154 protein selected from the groupconsisting of SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, and/or SEQ IDNO:78 and/or is a protein with CD154 activity, said IL-5 protein elicitsan immune response against a IL-5 protein selected from the groupconsisting of SEQ ID NO:81 and/or SEQ ID NO:86 and/or is a protein withIL-5 activity, said IL-13 protein elicits an immune response against anIL-13 protein selected from the group consisting of SEQ ID NO:92, SEQ IDNO:97, SEQ ID NO:100, and/or SEQ ID NO:105 and/or is a protein withIL-13 activity, said interferon alpha protein elicits an immune responseagainst an interferon alpha protein selected from the group consistingof SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ IDNO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168,and/or SEQ ID NO:171 and/or is a protein with interferon alpha activity,and/or said GMCSF protein elicits an immune response against a GMCSFprotein selected from the group consisting of SEQ ID NO:120 and/or SEQID NO:125 and/or is a protein with GM-CSF activity.

The present invention also includes methods to produce any of theproteins of the present invention using nucleic acid molecules of thepresent invention and recombinantly using such nucleic acid molecules.

The present invention also includes an isolated protein selected fromthe group consisting of: (a) (i) an isolated protein of at least about20 amino acids in length, wherein said protein is encoded by a nucleicacid molecule, wherein said nucleic acid molecule has an at least60contiguous nucleotide region identical in sequence to a 60 contiguousnucleotide region of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:4, and/or SEQ ID NO:19; and/or (ii)an isolated protein of at least about 20 amino acids in length, whereinsaid protein has an at least 20 contiguous amino acid region identicalin sequence to a 20 contiguous amino acid region selected from the groupconsisting of SEQ ID NO:2 and/or SEQ ID NO:20, wherein said isolatedprotein elicits an immune response against a canine IL-4 protein and/orhas IL-4 activity; (b) (i) an isolated protein of at least about 20amino acids in length, wherein said protein is encoded by a nucleic acidmolecule, wherein said nucleic acid molecule has an at least60contiguous nucleotide region identical in sequence to a 60 contiguousnucleotide region of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:22, SEQ ID NO:25, SEQID NO:28, SEQ ID NO:30, SEQ ID NO:33, and/or SEQ ID NO:36; and/or (ii)an isolated protein of at least about 20 amino acids in length, whereinsaid protein has an at least 20 contiguous amino acid region identicalin sequence to a 20 contiguous amino acid region selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31,and/or SEQ ID NO:34, wherein said isolated protein is capable ofeliciting an immune response against a canine Flt-3 ligand proteinand/or has Flt-3 activity; (c) (i) an isolated protein of at least about20 amino acids in length, wherein said protein is encoded by a nucleicacid molecule, wherein said nucleic acid molecule has an at least60contiguous nucleotide region identical in sequence to a 60 contiguousnucleotide region of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46,and/or SEQ ID NO:48; and/or (ii) an isolated protein of at least about20 amino acids in length, wherein said protein has an at least 20contiguous amino acid region identical in sequence to a 20 contiguousamino acid region selected from the group consisting of SEQ ID NO:44and/or SEQ ID NO:49, wherein said isolated protein is capable ofeliciting an immune response against a feline Flt-3 ligand proteinand/or has Flt-3 activity; (d)(i) an isolated protein of at least about30 amino acids in length, wherein said protein is encoded by a nucleicacid molecule, wherein said nucleic acid molecule has an at least90contiguous nucleotide region identical in sequences to a 90 contiguousnucleotide region of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:55, and/or SEQ IDNO:57; and/or (ii) an isolated protein of at least about 30 amino acidsin length, wherein said protein has an at least 30 contiguous amino acidregion identical in sequence to a 30 contiguous amino acid regionselected from the group consisting of SEQ ID NO:53, SEQ ID NO:58,wherein said isolated protein is capable of eliciting an immune responseagainst a canine CD40 protein and/or has CD40 activity; (e) (i) anisolated protein of at least about 20 amino acids in length, whereinsaid protein is encoded by a nucleic acid molecule, wherein said nucleicacid molecule has an at least60 contiguous nucleotide region identicalin sequence to a 60 contiguous nucleotide region of a nucleic acidsequence comprising SEQ ID NO:60; and/or (ii) an isolated protein of atleast about 20 amino acids in length, wherein said protein has an atleast 20 contiguous amino acid region identical in sequence to a 20contiguous amino acid region comprising the amino acid sequence SEQ IDNO:61, wherein said isolated protein is capable of eliciting an immuneresponse against a feline CD40protein and/or has CD40 activity; (f)(i)an isolated protein of at least about 35 amino acids in length, whereinsaid protein is encoded by a nucleic acid molecule, wherein said nucleicacid molecule has an at least105 contiguous nucleotide region identicalin sequence to a 105 contiguous nucleotide region of a nucleic acidsequence selected from the group consisting of SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:67, and/or SEQ ID NO:69; and/or (ii) an isolatedprotein of at least about 35 amino acids in length, wherein said proteinhas an at least 35 contiguous amino acid region identical in sequence toa 35 contiguous amino acid region selected from the group consisting ofSEQ ID NO:65 and/or SEQ ID NO:70, wherein said isolated protein iscapable of eliciting an immune response against a canine CD154 proteinand/or has CD154 activity; (g)(i) an isolated protein of at least about50 amino acids in length, wherein said protein is encoded by a nucleicacid molecule, wherein said nucleic acid molecule has an at least150contiguous nucleotide region identical in sequence to a 150 contiguousnucleotide region of a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:72, SEQ ID NO:75, and/or SEQ ID NO:77; and/or(ii) an isolated protein of at least about 50 amino acids in length,wherein said protein has an at least 50 contiguous amino acid regionidentical in sequence to a 50 contiguous amino acid region selected fromthe group consisting of SEQ ID NO:73 and/or SEQ ID NO:78, wherein saidisolated protein is capable of eliciting an immune response against afeline CD154 protein and/or has CD154 activity; (h)(i) an isolatedprotein of at least about 20 amino acids in length, wherein said proteinis encoded by a nucleic acid molecule, wherein said nucleic acidmolecule has an at least60 contiguous nucleotide region identical insequence to a 60 contiguous nucleotide region of a nucleic acid sequenceselected from the group consisting of SEQ ID NO:80, SEQ ID NO:83, and/orSEQ ID NO:85; and/or (ii) an isolated protein of at least about 20 aminoacids in length, wherein said protein has an at least 20 contiguousamino acid region identical in sequence to a 20 contiguous amino acidregion selected from the group consisting of SEQ ID NO:81 and/or SEQ IDNO:86, wherein said isolated protein is capable of eliciting an immuneresponse against a canine IL-5 protein and/or has IL-5 activity; (i)(i)an isolated protein of at least about 15 amino acids in length, whereinsaid protein is encoded by a nucleic acid molecule, wherein said nucleicacid molecule has an at least45 contiguous nucleotide region identicalin sequence to a 45 contiguous nucleotide region of a nucleic acidsequence selected from the group consisting of SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:99, SEQ ID NO:102, and/or SEQ ID NO:104; and/or (ii) an isolatedprotein of at least about 15 amino acids in length, wherein said proteinhas an at least 15 contiguous amino acid region identical in sequence toa 15 contiguous amino acid region selected from the group consisting ofSEQ ID NO:92, SEQ ID NO:97, SEQ ID NO:100, and/or SEQ ID NO:105, whereinsaid isolated protein is capable of eliciting an immune response againsta canine IL-13 protein and/or has IL-13 activity; (j) (i) an isolatedprotein encoded by a nucleic acid molecule selected from the groupconsisting of SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ IDNO:116, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQID NO:167, and/or SEQ ID NO:170, and/or (ii) an isolated proteinselected from the group consisting of SEQ ID NO:108, SEQ ID NO:111, SEQID NO:114, SEQ ID NO:117, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162,SEQ ID NO:165, SEQ ID NO:168, and/or SEQ ID NO:171, wherein saidisolated protein is capable of eliciting an immune response against afeline interferon alpha protein and/or has interferon alpha activity;(k) (i) an isolated protein encoded by a nucleic acid molecule selectedfrom the group consisting of SEQ ID NO:119, SEQ ID NO:122, and/or SEQ IDNO:124, and/or (ii) an isolated protein selected from the groupconsisting of SEQ ID NO:120 and/or SEQ ID NO:125, wherein said isolatedprotein is capable of eliciting an immune response against a felineGM-CSF and/or has GM-CSF activity.

The present invention also includes an isolated protein selected fromthe group consisting of: (a) a protein having an amino acid sequencethat is at least about 85 percent identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:2 and/or SEQ ID NO:20;(b) a protein having an amino acid sequence that is at least about 75percent identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31,and/or SEQ ID NO:34; (c) a protein having an amino acid sequence that isat least about 75 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:44 and/or SEQ ID NO:49; (d) aprotein having an amino acid sequence that is at least about 70 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:53 and/or SEQ ID NO:58; (e) a protein having an amino acidsequence that is at least about 60 percent identical to an amino acidsequence comprising SEQ ID NO:61; (f) a protein having an amino acidsequence that is at least about 80 percent identical to an amino acidsequence selected from the group consisting of SEQ ID NO:65 and/or SEQID NO:70; (g) a protein having an amino acid sequence that is at leastabout 85 percent identical to the amino acid sequence SEQ ID NO:73and/or SEQ ID NO:78; (h) a protein having an amino acid sequence that isat least about 85 percent identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NO:81 and/or SEQ ID NO:86; (i) aprotein having an amino acid sequence that is at least about 70 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO:100, and/or SEQ ID NO:105; (j)a protein having an amino acid sequence selected from the groupconsisting of SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ IDNO:117, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQID NO:168, and/or SEQ ID NO:171, and/or (k) a protein having an aminoacid sequence selected from the group consisting of SEQ ID NO:120,and/or SEQ ID NO:125.

The present invention also includes isolated antibodies that selectivelybind to a protein of the present invention.

One aspect of the present invention is a therapeutic composition that,when administered to an animal, regulates an immune response in saidanimal, said therapeutic composition comprising a therapeutic compoundselected from the group consisting of: an immunoregulatory protein ofthe present invention; a mimetope of any of said immunoregulatoryproteins; and a multimeric form of any of said immunoregulatoryproteins; an isolated nucleic acid molecule of the present invention; anantibody that selectively binds to any of said immunoregulatoryproteins; and/or an inhibitor of a immunoregulatory protein activityidentified by its ability to inhibit the activity of any of saidimmunoregulatory proteins. Yet another aspect of the present inventionis a method to regulate an immune response in an animal comprisingadministering to the animal a therapeutic composition of the presentinvention.

The present invention also includes a method to produce animmunoregulatory protein, said method comprising culturing a cellcapable of expressing said protein, said protein being encoded by anucleic acid molecule of the present invention.

One embodiment of the present invention is a method to identify acompound capable of regulating an immune response in an animal, saidmethod comprising: (a) contacting an isolated canine IL-4 protein of thepresent invention with a putative inhibitory compound under conditionsin which, in the absence of said compound, said protein has T cellproliferation stimulating activity; and determining if said putativeinhibitory compound inhibits said activity; (b) contacting an isolatedcanine Flt-3 ligand protein of the present invention with a putativeinhibitory compound under conditions in which, in the absence of saidcompound, said protein has dendritic precursor cell proliferationstimulating activity; and determining if said putative inhibitorycompound inhibits said activity; (c) contacting an isolated feline Flt-3ligand protein of the present invention with a putative inhibitorycompound under conditions in which, in the absence of said compound,said protein has dendritic precursor cell proliferation stimulatingactivity; and determining if said putative inhibitory compound inhibitssaid activity; (d) contacting an isolated canine CD40 protein of thepresent invention with a putative inhibitory compound under conditionsin which, in the absence of said compound, said protein has CD40 ligandbinding activity; and determining if said putative inhibitory compoundinhibits said activity; (e) contacting an isolated feline CD40 proteinof the present invention with a putative inhibitory compound underconditions in which, in the absence of said compound, said protein hasCD40 ligand biding activity; and determining if said putative inhibitorycompound inhibits said activity; (f) contacting an isolated canine CD154protein of the present invention with a putative inhibitory compoundunder conditions in which, in the absence of said compound, said proteinhas B cell proliferation activity; and determining if said putativeinhibitory compound inhibits said activity; (g) contacting an isolatedfeline CD154 protein of the present invention with a putative inhibitorycompound under conditions in which, in the absence of said compound,said protein has B cell proliferation activity; and determining if saidputative inhibitory compound inhibits said activity; (h) contacting anisolated canine IL-5 protein of the present invention with a putativeinhibitory compound under conditions in which, in the absence of saidcompound, said protein has TF-1 cell proliferation activity; anddetermining if said putative inhibitory compound inhibits said activity;(i) contacting an isolated canine IL-13 protein of the present inventionwith a putative inhibitory compound under conditions in which, in theabsence of said compound, said protein has TF-1 cell proliferationactivity; and determining if said putative inhibitory compound inhibitssaid activity; (j) contacting an isolated feline IFNα protein of thepresent invention with a putative inhibitory compound under conditionsin which, in the absence of said compound, said protein has inhibitionof proliferation of GM-CSF stimulated TF-1 cell activity; anddetermining if said putative inhibitory compound inhibits said activity;or (k) contacting an isolated feline GMCSF protein of the presentinvention with a putative inhibitory compound under conditions in which,in the absence of said compound, said protein has TF-1 cellproliferation activity; and determining if said putative inhibitorycompound inhibits said activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for isolated canine interleukin-4, canineor feline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF proteins, isolated canine interleukin-4, canine or felineFlt-3 ligand, canine or feline CD40, canine or feline CD154, canineinterleukin-5, canine interleukin-13, feline interferon alpha, or felineGM-CSF nucleic acid molecules, antibodies directed against canineinterleukin-4, canine or feline Flt-3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF proteins, and compoundsderived therefrom that regulate the immune response of an animal (e.g.inhibitors, antibodies and peptides).

Canine IL-4 protein can refer to a canine IL-4 protein, includinghomologs thereof. Canine Flt-3 ligand protein can refer to a canineFlt-3 ligand, including homologs thereof, and feline Flt-3 ligand canrefer to feline Flt-3 ligand, including homologs thereof. Canine CD40can refer to a canine CD4-, including homologs thereof; feline CD40 canrefer to a feline CD40, including homologs thereof. Canine CD154 canrefer to a canine CD154, including homo logs thereof; feline CD154 canrefer to a feline CD154, including homolog thereof. Canine IL-5 canrefer to canine IL-5, including homologs thereof; canine IL-13 can referto canine IL-13, including homologs thereof. Feline IFNα can refer to afeline IFNα, including homologs thereof, and feline GM-CSF can refer toa feline GM-CSF, including homologs thereof. As used herein, the phrase“regulate an immune response” refers to modulating the activity of cellsor molecules involved in an immune response. The term “regulate” canrefer to increasing or decreasing an immune response. Regulation of animmune response can be determined using methods known in the art as wellas methods disclosed herein. The term, “immunoregulatory protein” refersto a protein that can modulate the activity of cells or of moleculesinvolved in an immune response. An immunoregulatory protein of thepresent invention refers to a canine IL-4, a canine and/or feline CD40,a canine and/or feline Flt3 ligand, a canine and/or feline CD154, acanine IL-5, a canine IL-13, a feline IFNα and/or a feline GM-CSFprotein as described herein. As used herein, the terms isolated canineinterleukin-4, canine or feline Flt-3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF proteins and/or isolatedcanine interleukin-4, canine or feline Flt-3 ligand, canine or felineCD40, canine or feline CD1 54, canine interleukin-5, canineinterleukin-13, feline interferon alpha, or feline GM-CSF nucleic acidmolecules refer to canine interleukin-4, canine or feline Flt-3 ligand,canine or feline CD40, canine or feline CD154, canine interleukin-5,canine interleukin-13, feline interferon alpha, or feline GM-CSFproteins and/or canine interleukin-4, canine or feline Flt-3 ligand,canine or feline CD40, canine or feline CD154, canine interleukin-5,canine interleukin-13, feline interferon alpha, or feline GM-CSF nucleicacid molecules derived from mammals and, as such, can be obtained fromtheir natural source, or can be produced using, for example, recombinantnucleic acid technology or chemical synthesis. Also included in thepresent invention is the use of these proteins, nucleic acid molecules,antibodies, and/or compounds derived therefrom as therapeuticcompositions to regulate the immune response of an animal as well as inother applications, such as those disclosed below.

One embodiment of the present invention is an isolated protein thatincludes a canine IL-4 protein, a canine and/or feline Flt-3 ligandprotein, a canine and/or feline CD40 protein, a canine and/or felineCD154 protein, a canine interleukin-5 protein, a canine interleukin-13protein, a feline interferon alpha protein, and/or a feline GM-CSFprotein. It is to be noted that the term “a” or “an” entity refers toone or more of that entity; for example, a protein refers to one or moreproteins or at least one protein. As such, the terms “a” (or “an”) ,“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably. According to the presentinvention, an isolated, or biologically pure, protein, is a protein thathas been removed from its natural milieu. As such, “isolated” and/or“biologically pure” do not necessarily reflect the extent to which theprotein has been purified. An isolated protein of the present inventioncan be obtained from its natural source, can be produced usingrecombinant DNA technology, or can be produced by chemical synthesis.Nucleic acid molecules of the present invention of known length isolatedfrom Canis familiaris are denoted as follows: IL-4 is denoted asnCaIL-4_(x), for example, nCaIL-4₅₄₉, wherein “#” refers to the numberof nucleotides in that molecule; and in a similar fashion, Flt-3 ligandnucleic acid molecules are referred to as nCaFlt3L_(x); CD40,nCaCD40_(x); CD154, nCaCD154_(x); IL-5, nCaIL-5_(x); and IL-13,nCaIL-13_(x). In a similar fashion, Flt-3 ligand nucleic acid moleculesof the present invention of known length isolated from Felis catus aredenoted as nFeFlt3L_(x), CD40, nFeCD40_(x); CD154, nFeCD154_(x); IFNα,nFeIFNα_(x); and GM-CSF (also denoted GMCSF), nFeGM-CSF_(x). Similarly,proteins of the present invention of known length isolated from Feliscatus are denoted as PFeFlt31_(x); PFeCD40_(x); PFeCD154_(x),PFeIFNα_(x), and/or PFeGM-CSF_(x); and proteins of the present inventionof known length isolated from Canis familiaris are denoted PCaIL-4_(x),PCaFlt3L_(x), PCaCD40_(x), PCaCD154_(x), PCaIL-5_(x), and/orPCaIL-13_(x).

As used herein, an isolated canine interleukin-4, canine or feline Flt-3ligand, canine or feline CD40, canine or feline CD154, canineinterleukin-5, canine interleukin-13, feline interferon alpha, and/orfeline GM-CSF ligand protein of the present invention (i.e., an canineinterleukin-4, canine or feline Flt-3 ligand canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF protein, respectively) can bea full-length protein or any homolog of such a protein. An isolated IL-4protein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response against, (or to) an IL-4 protein, bind to an IL-4receptor, stimulate B cell differentiation or activation or stimulateproduction of immunoglobulin by a B cell. An isolated Flt-3 ligandprotein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response against a Flt-3 ligand protein, bind to Flt-3 receptoror stimulate Flt-3 receptor-bearing hematopoietic stem cells, earlyhematopoietic progenitor cells or immature lymphocytes. An isolated CD40protein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response against a CD40 protein, bind to CD154 or stimulate CD154-bearing B cells, T-cells, and/or epithelial cells. An isolated CD154protein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response to a CD154 protein, bind to CD40 or stimulateCD40-bearing B cells, T cells, and/or epithelial cells. An isolated IL-5protein of the present invention, including a homolog, can be identifiedin a straight-forward manner by the protein's ability to elicit animmune response to an IL-5 protein, bind to an IL-5 receptor, and/orstimulate eosinophils and/or cause thymocytes to produce cyctotoxic Tcells. An isolated IL-13 protein of the present invention, including ahomolog, can be identified in a straight-forward maner by the protein'sability to elicit an immune response to an IL-13 protein, bind to anIL-13 receptor, and/or stimulate B cells, up-regulate expression of MHCclass II and/or CD23 on monocytes, macrophages and/or B cells; and/orinhibition of proinflammatory cytokines. An isolated interferon alphaprotein of the present invention, including a homolog, an be identifiedin a straight-forward manner by the protein's ability to elicit animmune response to an interferon alpha protein, bind to an interferonalpha receptor, and/or activate NK cells and/or inhibit viralreplication. An isolated GM-CSF proteins of the present invention,including a homolog, can be identified in a straight-forward manner bythe protein's ability to elicit an immune response to a GM-CSF protein,bind to a GM-CSF receptor, and/or activate granulocytes and/ormacrophages. Examples of protein homologs of the present inventioninclude immunoregulatory proteins of the present invention in whichamino acids have been deleted (e.g., a truncated version of the protein,such as a peptide), inserted, inverted, substituted and/or derivatized(e.g., by glycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitoylation, amidation and/or addition ofglycerophosphatidyl inositol) such that the protein homolog includes atleast one epitope capable of eliciting an immune response against theparent protein, of binding to an antibody directed against the parentprotein and/or of binding to the parent's receptor, where the termparent refers to the longer and/or full-length protein that the homologis derived from. That is, when the homolog is administered to an animalas an immunogen, using techniques known to those skilled in the art,that animal will produce an immune response against at least one epitopeof an immunoregulatory protein of the present invention, depending uponwhich protein is administered to an animal. The ability of a protein toeffect an immune response can be measured using techniques known tothose skilled in the art. As used herein, the term “epitope” refers tothe smallest portion of a protein capable of selectively binding to theantigen binding site of an antibody. It is well accepted by thoseskilled in the art that the minimal size of a protein epitope capable ofselectively binding to the antigen binding site of an antibody is aboutfive or six to seven amino acids.

Homologs of immunoregulatory proteins of the present invention can bethe result of natural allelic variation, including natural mutation.Protein homologs of the present invention can also be produced usingtechniques known in the art including, but not limited to, directmodifications to the protein and/or modifications to the gene encodingthe protein using, for example, classic or recombinant DNA techniques toeffect random or targeted mutagenesis.

Immunoregulatory proteins of the present invention include variants of afull-length protein of a protein of the present invention. Such variantsinclude proteins that are less than full-length. As used herein, variantof the present invention refer to nucleic acid molecules that arenaturally-occurring as defined below, and may result from alternativeRNA splicing, alternative termination of an amino acid sequence or DNArecombination. Examples of variants include allelic variants as definedbelow. It is to be noted that a variant is an example of a homolog ofthe present invention.

Immunoregulatory proteins of the present invention are encoded bynucleic acid molecules of the present invention. As used herein, an IL-4nucleic acid molecule includes nucleic acid sequences related to anatural canine IL-4 gene. As used herein, a Flt-3 ligand nucleic acidmolecule includes nucleic acid sequences related to a natural canineFlt-3 ligand gene. As used herein, a CD40 nucleic acid molecule includesnucleic acid sequences related to a natural CD40 gene. As used herein, aCD154 nucleic acid molecule includes nucleic acid sequences related to anatural CD154 gene. As used herein, an IL-5 nucleic acid moleculeincludes nucleic acid sequences related to a natural IL-5 gene. As usedherein, an IL-13 nucleic acid molecule includes nucleic acid sequencesrelated to a natural IL-13 gene. As used herein, an IFNα nucleic acidmolecule includes nucleic acid sequences related to a natural IFNα gene.As used herein, a GM-CSF nucleic acid molecule includes nucleic acidsequences related to a natural GM-CSF gene. As used herein, a canineIL-4, a canine and/or feline CD40, a canine and/or feline Flt3 ligand, acanine and/or feline CD154, a canine IL-5, a canine IL-13, a felineIFNα, and/or a feline GM-CSF gene refers to the natural genomic elementsthat encode an canine IL-4, a canine and/or feline CD40, a canine and/orfeline Flt3 ligand, a canine and/or feline CD154, a canine IL-5, acanine IL-13, a feline IFNα, and/or a feline GM-CSF proteins,respectively, and includes all regions such as regulatory regions thatcontrol production of the protein encoded by the gene (such as, but notlimited to, transcription, translation or post-translation controlregions) as well as the coding region itself, and any introns ornon-translated coding regions. As used herein, a gene that “includes” or“comprises” a sequence may include that sequence in one contiguousarray, or may include the sequence as fragmented exons. As used herein,the term “coding region” refers to a continuous linear array ofnucleotides that translates into a protein. A full-length coding regionis that region that is translated into a full-length, i.e., a complete,protein as would be initially translated in its natural milieu, prior toany post-translational modifications.

In one embodiment, an IL-4 gene of the present invention includes thenucleic acid sequence SEQ ID NO:1, as well as the complement of SEQ IDNO:1. Nucleic acid sequence SEQ ID NO:1 represents the deduced sequenceof the coding strand of a cDNA (complementary DNA) denoted herein asnucleic acid molecule nCaIL-4₅₄₉, the production of which is disclosedin the Examples. Nucleic acid molecule nCaIL-4₅₄₉ comprises anapparently full-length coding region of canine IL-4. The complement ofSEQ ID NO:1 (represented herein by SEQ ID NO:3) refers to the nucleicacid sequence of the strand fully complementary to the strand having SEQID NO:1, which can easily be determined by those skilled in the art.Likewise, a nucleic acid sequence complement of any nucleic acidsequence of the present invention refers to the nucleic acid sequence ofthe nucleic acid strand that is fully complementary to (i.e., can form adouble helix with) the strand for which the sequence is cited. It shouldbe noted that since nucleic acid sequencing technology is not entirelyerror-free, SEQ ID NO:1 (as well as other nucleic acid and proteinsequences presented herein) represents an apparent nucleic acid sequenceof the nucleic acid molecule encoding an immunoregulatory protein of thepresent invention.

In another embodiment, a Flt-3 ligand gene of the present inventionincludes the nucleic acid sequence SEQ ID NO:6, as well as thecomplement represented by SEQ ID NO:8. Nucleic acid sequence SEQ ID NO:6represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nCaFlt3L₁₀₁₃, the production of which isdisclosed in the Examples. Nucleic acid molecule nCaFlt3L₁₀₁₃ comprisesan apparently full-length coding region of canine Flt-3 ligand.

In another embodiment, a Flt-3 ligand gene of the present inventionincludes the nucleic acid sequence SEQ ID NO:43, as well as thecomplement represented by SEQ ID NO:45. Nucleic acid sequence SEQ IDNO:43 represents the deduced sequence of the coding strand of a cDNAdenoted herein as nucleic acid molecule nFeFlt3L₉₄₂, the production ofwhich is disclosed in the Examples. Nucleic acid molecule nFeFlt3L₉₄₂comprises an apparently full-length coding region of feline Flt-3ligand.

In another embodiment, a CD40 gene of the present invention includes thenucleic acid sequence SEQ ID NO:52, as well as the complementrepresented by SEQ ID NO:54. Nucleic acid sequence SEQ ID NO:52represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nCaCD40₁₄₂₅, the production of which isdisclosed in the Examples. Nucleic acid molecule nCaCD40₁₄₂₅ comprisesan apparently full-length coding region of canine CD40.

In another embodiment, a CD40 gene of the present invention includes thenucleic acid sequence SEQ ID NO:60, as well as the complementrepresented by SEQ ID NO:62. Nucleic acid sequence SEQ ID NO:60represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nFeCD40₃₃₆, the production of which isdisclosed in the Examples. Nucleic acid molecule nFeCD40₃₃₆ comprises anapparent portion of the coding region of feline CD40.

In another embodiment, a CD154 gene of the present invention includesthe nucleic acid sequence SEQ ID NO:64, as well as the complementrepresented by SEQ ID NO:66. Nucleic acid sequence SEQ ID NO:64represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nCaCD154₁₈₇₈, the production of which isdisclosed in the Examples. Nucleic acid molecule nCaCD154₁₈₇₈ comprisesan apparently full-length coding region of canine CD154.

In another embodiment, a CD154 gene of the present invention includesthe nucleic acid sequence SEQ ID NO:72, as well as the complementrepresented by SEQ ID NO:74. Nucleic acid sequence SEQ ID NO:72represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nFeCD154₈₈₅, the production of which isdisclosed in the Examples. Nucleic acid molecule nFeCD154₈₈₅ comprisesan apparently full-length coding region of feline CD154.

In another embodiment, an IL-5 gene of the present invention includesthe nucleic acid sequence SEQ ID NO:80, as well as the complementrepresented by SEQ ID NO:82. Nucleic acid sequence SEQ ID NO:80represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nCaIL-5₆₁₀, the production of which isdisclosed in the Examples. Nucleic acid molecule nCaIL-5₆₁₀ comprises anapparently full-length coding region of canine IL-5.

In another embodiment, an IL-13 gene of the present invention includesthe nucleic acid sequence SEQ ID NO:91, as well as the complementrepresented by SEQ ID NO:93. Nucleic acid sequence SEQ ID NO:91represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nCaIL-13₁₃₀₂, the production of which isdisclosed in the Examples. Nucleic acid molecule nCaIL-13₁₃₀₂ comprisesan apparently full-length coding region of canine IL-13.

In another embodiment, an IFNα gene of the present invention includesthe nucleic acid sequence SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:155,SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ ID NO:167, or SEQ IDNO:170, as well as the complement represented by, respectively, SEQ IDNO:109, SEQ ID NO:112, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:163, orSEQ ID NO:166, SEQ ID NO:169, and SEQ ID NO:172. Nucleic acid sequencesSEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:155, SEQ ID NO:158, SEQ IDNO:161, SEQ ID NO:164, SEQ ID NO:167, and SEQ ID NO:170 represent thededuced sequences of the coding strands of cDNAs denoted herein asnucleic acid molecules nFeIFNα_(567a), nFeIFNα_(567b), nFeIFNα_(567c),nFeIFNα_(498c), nFeIFNα_(582d), nFeIFNα_(513d), nFeIFNα_(567e) andnFeIFNα_(498e), respectively. Each of these nucleic acid molecules, theproduction of which is disclosed in the Examples, comprises anapparently full-length coding region of a feline IFNα protein.

In another embodiment, a GM-CSF gene of the present invention includesthe nucleic acid sequence SEQ ID NO:119, as well as the complementrepresented by SEQ ID NO:121. Nucleic acid sequence SEQ ID NO:119represents the deduced sequence of the coding strand of a cDNA denotedherein as nucleic acid molecule nFeGM-CSF₄₄₄, the production of which isdisclosed in the Examples. Nucleic acid molecule nFeGM-CSF₄₄₄ comprisesan apparently full-length coding region of feline GM-CSF.

Additional immunoregulatory nucleic acid molecules and proteins of thepresent invention having specific sequence identifiers are described inTable 1.

TABLE 1 Sequence identification numbers (SEQ ID NOs) and theircorresponding nucleic acid molecules or proteins. SEQ ID NO: DESCRIPTION1 nCaIL-4₅₄₉ coding strand 2 PCaIL-4₁₃₂ 3 nCaIL-4₅₄₉ complementarystrand 4 nCaIL-4₃₉₆ coding strand 5 nCaIL-4₃₉₆ complementary strand 6nCaFlt3L₁₀₁₃ coding strand 7 PCaFlt3L₂₉₄ 8 nCaFlt3L₁₀₁₃ complementarystrand 9 nCaFlt3L₈₈₂ coding strand 10 nCaFlt3L₈₈₂ complementary strand19 nCaIL-4₃₂₄ coding strand 20 PCaIL-4₁₀₈ 21 nCaIL-₃₂₄ complementarystrand 22 nCaFlt3L₈₀₄ coding strand 23 PCaFlt3L₂₆₈ 24 nCaFlt3L₈₀₄complementary strand 25 nCaFlt3L₉₈₅ coding strand 26 PCaFlt3L₂₇₆ 27nCaFlt3L₉₈₅ complementary strand 28 nCaFlt3L₈₂₈ coding strand 29nCaFlt3L₈₂₈ complementary strand 30 nCaFlt3L₇₅₀ coding strand 31PCaFlt3L₂₅₀ 32 nCaFlt3L₇₅₀ complementary strand 33 nCaFlt3L₁₀₁₉ codingstrand 34 PCaFlt3L₃₁ 35 nCaFlt3L₁₀₁₉ complementary strand 36 nCaFlt3L₉₃coding strand 37 nCaFlt3L₉₃ complementary strand 41 nFeFlt3L₃₉₅ codingstrand 42 nFeFlt3L₇₉₃ coding strand 43 nFeFlt3L₉₄₂ coding strand 44PFeFlt3L₂₉₁ 45 nFeFlt3L₉₄₂ complementary strand 46 nFeFlt3L₈₇₃ codingstrand 47 nFeFlt3L₈₇₃ complementary strand 48 nFeFlt3L₇₉₅ coding strand49 PFeFlt3L₂₆₅ 50 nFeFlt3L₇₉₅ complementary strand 51 nCaCD40₃₂₁ codingstrand 52 nCaCD40₁₄₂₅ coding strand 53 PCaCD40₂₇₄ 54 nCaCD40₁₄₂₅complementary strand 55 nCaCD40₈₂₂ coding strand 56 nCaCD40₈₂₂complementary strand 57 nCaCD40₇₆₅ coding strand 58 PCaCD40₂₅₅ 59nCaCD40₇₆₅ complementary strand 60 nFeCD40₃₃₆ coding strand 61PFeCD40₁₁₂ 62 nFeCD40₃₃₆ complementary strand 63 nCaCD154₃₉₀ codingstrand 64 nCaCD154₁₈₇₈ coding strand 65 PCaCD154₂₆₀ 66 nCaCD154₁₈₇₈complementary strand 67 nCaCD154₇₈₀ coding strand 68 nCaCD154₇₈₀complementary strand 69 nCaCD154₆₃₃ coding strand 70 PCaCD154₂₁₁ 71nCaCD154₆₃₃ complementary strand 72 nFeCD154₈₈₅ coding strand 73PFeCD154₂₆₀ 74 nFeCD154₈₈₅ complementary strand 75 nFeCD154₇₈₀ codingstrand 76 nFeCD154₇₈₀ complementary strand 77 nFeCD154₆₃₃ coding strand78 PFeCD154₂₁₁ 79 nFeCD154₆₃₃ complementary strand 80 nCaIL-5₆₁₀ codingstrand 81 PCaIL-5₁₃₄ 82 nCaIL-5₆₁₀ complementary strand 83 nCaIL-5₄₀₂coding strand 84 nIL-5₄₀₂ complementary strand 85 nCaIL-5₃₄₅ codingstrand 86 PCaIL-5₁₁₅ 87 nCaIL-5₃₄₅ complementary strand 88 nCaIL-13₁₆₆coding strand 89 nCaIL-13₂₇₂ coding strand 90 nCaIL-13₂₇₈ coding strand91 nCaIL-13₁₃₀₂ coding strand 92 PCaIL-13₁₃₁ 93 nCaIL-13₁₃₀₂complementary strand 94 nCaIL-13₃₉₃ coding strand 95 nCaIL-13₃₉₃complementary strand 96 nCaIL-13₃₃₃ coding strand 97 PaIL-13₁₁₁ 98nCaIL-13₃₃₃ complementary strand 99 nCaIL-13₁₂₆₉ coding strand 100PCaIL-13₁₃₀ 101 nCaIL-13₁₂₆₉ complementary strand 102 nCaIL-13₃₉₀ codingstrand 103 nCaIL-13₃₉₀ complementary strand 104 nCaIL-13₃₃₀ codingstrand 105 PCaIL-13₁₁₀ 106 nCaIL-13₃₃₀ complementary strand 107nFeIFNα_(567a) coding strand 108 PFeIFNα_(189a) 109 nFeIFNα_(567a)complementary strand 110 nFeIFNα_(567b) coding strand 111 PFeIFNα_(189b)112 nFeIFNα_(567b) complementary strand 113 nFeIFNα_(498a) coding strand114 PFeIFNα_(166a) 115 nFeIFNα_(498a) complementary strand 116nFeFeIFNα_(498b) coding strand 117 PFeIFNα_(166b) 118 nFeIFNα_(498b)complementary strand 119 nFeGMCSF₄₄₄ coding strand 120 PFeGMCSF₁₄₄ 121nFeGMCSF₄₄₄ complementary strand 122 nFeGMCSF₄₃₂ coding strand 123nFeGMCSF₄₃₂ complementary strand 124 nFeGMCSF₃₈₁ coding strand 125PFeGMCSF₁₂₇ 126 nFeGMCSF₃₈₁ complementary strand 155 nFeIFNα_(567c) 156PFeIFNα_(189c) 157 nFeIFNα_(567a) complementary strand 158nFeIFNα_(498c) 159 PFeIFNα_(166c) 160 nFeIFNα_(498c) complementarystrand 161 nFeIFNα_(582d) 162 PFeIFNα_(194d) 163 nFeIFNα_(582d)complementary strand 164 nFeIFNα_(513d) 165 PFeIFNα_(171d) 166nFeIFNα_(513d) complementary strand 167 nFeIFNα_(567e) 168PFeIFNα_(189e) 169 nFeIFNα_(567e) complementary strand 170nFeIFNα_(498e) 171 PFeIFNα_(166e) 172 nFeIFNα_(498e) complementarystrand

In another embodiment, an IL-4 gene or nucleic acid molecule can be anallelic variant that includes a similar but not identical sequence toSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, SEQ IDNO:2 1, and/or any other IL-4 nucleic acid sequence cited herein. Inanother embodiment, a Flt-3 ligand gene or nucleic acid molecule can bean allelic variant that includes a similar but not identical sequence toSEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50 and/or any other Flt-3ligand nucleic acid sequence cited herein. In another embodiment, a CD40gene or nucleic acid molecule can be an allelic variant that includes asimilar but not identical sequence to SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:62 and/or other CD40 nucleic acid sequence citedherein. In another embodiment, a CD154 gene or nucleic acid molecule canbe an allelic variant that includes a similar but not identical sequenceto SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68,SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75,SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:79 and/or any other CD154 nucleicacid sequences cited herein. In another embodiment, an IL-5 gene ornucleic acid molecule can be an allelic variant that includes a similarbut not identical sequence to SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83,SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:87 and/or any other IL-5 nucleicacid sequence cited herein. In another embodiment, an IL-13 gene ornucleic acid molecule can be an allelic variant that includes a similarbut not identical sequence to SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90,SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96,SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103,SEQ ID NO:104, SEQ ID NO:106 and/or any other IL-13 nucleic acidsequence cited herein. In another embodiment, an IFNα gene or nucleicacid molecule can be an allelic variant that includes a similar but notidentical sequence to SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:167, SEQID NO:169, SEQ ID NO:170 and/or SEQ ID NO:172, and/or any other IFNαnucleic acid sequence cited herein. In another embodiment, a GM-CSF geneor nucleic acid molecule can be an allelic variant that includes asimilar but not identical sequence to SEQ ID NO:119, SEQ ID NO:121, SEQID NO:122, SEQ ID NO:123, SEQ ID NO:124, and/or SEQ ID NO:126 and/or anyother GM-CSF nucleic acid cited herein. An allelic variant of a canineinterleukin-4, canine or feline Flt-3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF gene, including the particularSEQ ID NO's cited herein, is a gene that occurs at essentially the samelocus (or loci) in the genome as the gene including the particular SEQID NO's cited herein, but which, due to natural variations caused by,for example, mutation or recombination, has a similar but not identicalsequence. Also includes in the term allelic variant are allelic variantsof cDNAs derived from such genes. Because natural selection typicallyselects against alterations that affect function, allelic variantsusually encode proteins having similar activity to that of the proteinencoded by the gene to which they are being compared. Allelic variantsof genes or nucleic acid molecules can also comprise alterations in the5′ or 3′ untranslated regions of the gene (e.g., in regulatory controlregions), or can involve alternative splicing of a nascent transcript,thereby bringing alterative exons into juxtaposition. Allelic variantsare well known to those skilled in the art and would be expected to befound within a given animal, since the respective genomes are diploid,and sexual reproduction will result in the reassortment of alleles.

The minimal size of an canine interleukin-4, canine or feline Flt-3ligand, canine or feline CD40, canine or feline CD154, canineinterleukin-5, canine interleukin-13, feline interferon alpha, or felineGM-CSF protein homolog of the present invention is a size sufficient tobe encoded by a nucleic acid molecule capable of forming a stable hybrid(i.e., hybridize under stringent hybridization conditions) with thecomplementary sequence of a nucleic acid molecule encoding thecorresponding natural protein. Stringent hybridization conditions aredetermined based on defined physical properties of the gene to which thenucleic acid molecule is being hybridized, and can be definedmathematically. Stringent hybridization conditions are thoseexperimental parameters that allow an individual skilled in the art toidentify significant similarities between heterologous nucleic acidmolecules. These conditions are well known to those skilled in the art.See, for example, Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al.,1984, Anal. Biochem. 138, 267-284, each of which is incorporated hereinby this reference. As explained in detail in the cited references, thedetermination of hybridization conditions involves the manipulation of aset of variables including the ionic strength (M, in moles/liter), thehybridization temperature (° C.), the concentration of nucleic acidhelix destabilizing agents, such as formamide, the average length of theshortest hybrid duplex (n), and the percent G+C composition of thefragment to which an unknown nucleic acid molecule is being hybridized.For nucleic acid molecules of at least about 150 nucleotides, thesevariables are inserted into a standard mathematical formula to calculatethe melting temperature, or T_(m), of a given nucleic acid molecule. Asdefined in the formula below, T_(m) is the temperature at which twocomplementary nucleic acid molecule strands will disassociate, assuming100% complementarity between the two strands:T_(m)=81.5° C.+16.6 log M+0.41(%G+C)−500/n−0.61(%formamide).For nucleic acid molecules smaller than about 50 nucleotides, hybridstability is defined by the dissociation temperature (T_(d)), which isdefined as the temperature at which 50% of the duplexes dissociate. Forthese smaller molecules, the stability at a standard ionic strength isdefined by the following equation:T_(d)=4(G+C)+2(A+T).A temperature of 5° C. below T_(d) is used to detect hybridizationbetween perfectly matched molecules.

Also well known to those skilled in the art is how base pair mismatch,i.e. differneces between two nucleic acid molecules being compared,including non-complementarity of bases at a given location, and gaps dueto insertion or deletion of one or more bases at a given location oneither of the nucleic acid molecules being compared, will affect T_(m)or T_(d) for nucleic acid molecules of different sizes. For example,T_(m) decreases about 1° C. for each 1% of mismatched base pairs forhybrids greater than about 150 bp, and T_(d) decreased about 5° C. foreach mismatched base pair for hybrids below about 50 bp. Conditions forhybrids between about 50 and about 150 base pairs can be determinedempirically and without undue experimentation using standard laboratoryprocedures well known to those skilled in the art. These simpleprocedures allow one skilled in the art to set the hybridizationconditions, by altering, for example, the salt concentration, theformamide concentration or the temperature, so that only nucleic acidhybrids with greater than a specified % base pair mismatch willhybridize. Stringent hybridization conditions are commonly understood bythose skilled in the art to be those experimental conditions that willallow about 30% base pair mismatch, i.e., about 70% identity. Becauseone skilled in the art can easily determine whether a given nucleic acidmolecule to be tested is less than or greater than about 50 nucleotides,and can therefore choose the appropriate formula for determininghybridization conditions, he or she can determine whether the nucleicacid molecule will hybridize with a given gene or specified nucleic acidmolecule under stringent hybridization conditions and similarly whetherthe nucleic acid molecule will hybridize under conditions designed toallow a desired amount of base pair mismatch.

Hybridization reactions are often carried out by attaching the nucleicacid molecule to be hybridized to a solid support such as a membrane,and then hybridizing with a labeled nucleic acid molecule, typicallyreferred to as a probe, suspended in a hybridization solution. Examplesof common hybridization reaction techniques include, but are not limitedto, the well-known Southern and northern blotting procedures. Typically,the actual hybridization reaction is done under non-stringentconditions, i.e., at a lower temperature and/or a higher saltconcentration, and then high stringency is achieved by washing themembrane in a solution with a higher temperature and/or lower saltconcentration in order to achieve the desired stringency.

Preferred portions, or fragments, of a canine interleukin-4, canine orfeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF, protein of the present invention include at least 15amino acids, at least 20 amino acids, at least 25 amino acids, at least30 amino acids, at least 35 amino acids, at least 40 amino acids, atleast 45 amino acids, at least 50 amino acids, at least 60 amino acids,at least 75 amino acids or at least 100 amino acids. An IL-4, IL-5,and/or IL-13 protein of the present invention can include at least aportion of an IL-4, IL-5, and/or IL-13 protein that is capable ofbinding to an IL-4, IL-5, and/or IL-13 receptor, respectively. IL4,IL-5, and IL-13 receptors are known to those of skill in the art, andare described in Janeway et al., in Immunobiology, the Immune System inHealth and Disease, Garland Publishing, Inc., N.Y., 1996 (which isincorporated herein by this reference in its entirety). The IL-4, IL-5,and/or IL-13 receptor-binding protein of an IL-4, IL-5, and/or IL-13protein, respectively, can be determined by incubating the protein withan isolated IL-4, IL-5, and/or IL-13 receptor, as appropriate, or a cellhaving an IL-4, IL-5, and/or IL-13 receptor on its surface, asappropriate. IL-4, IL-5, and/or IL-13 protein binding to purified IL-4,IL-5, and/or IL-13 receptor, respectively, can be determined usingmethods known in the art including Biacore® screening, confocalimmunofluorescent microscopy, immunoprecipitation, gel chromatography,determination of inhibition of binding of antibodies that bindspecifically to the IL-4, IL-5, and/or IL-13 binding domain of an IL-4,IL-5, and/or IL-13 receptor, ELISA using an IL-4, IL-5, and/or IL-13receptor, respectively, labeled with a detectable tag such as an enzymeor chemiluminescent tag or yeast-2 hybrid technology. A Flt-3 ligandprotein of the present invention can include at least a portion of aFlt-3 ligand protein that is capable of binding to Flt-3 receptor orstimulating Flt-3 receptor-bearing hematopoietic stem cells, earlyhematopoietic progenitor cells or immature lymphocytes. Flt-3 receptorsare known to those of skill in the art, and are described in Drexler,Leukemia, vol. 10, pp. 588-599, 1996 (which is incorporated herein inits entirety by this reference). The Flt-3 receptor-binding portion of aFlt-3 ligand protein can be determined by incubating the protein withisolated Flt-3 receptor or a cell having a Flt-3 receptor on itssurface. Flt-3 ligand protein binding to purified Flt-3 receptor can bedetermined using methods known in the art including Biacore® screening,confocal immunofluorescent microscopy, immunoprecipitation, gelchromatography, determination of inhibition of binding of antibodiesthat bind specifically to the Flt-3 ligand binding domain of a Flt-3receptor, ELISA using a Flt-3 receptor labeled with a detectable tagsuch as an enzyme or chemiluminescent tag or yeast-2 hybrid technology.A CD40 and/or CD154 protein of the present invention can include atleast a portion of a CD40 and/or CD154 protein that is capable ofbinding to a CD40 and/or CD154 receptor, respectively, or stimulatingCD40 and/or CD154 receptor-bearing hematopoietic stem cells, earlyhematopoietic progenitor cells or immature lymphocytes. The CD40 and/orCD154 receptor-binding portion of a CD40 and/or CD154 protein can bedetermined by incubating the protein with isolated CD40 and/or CD154receptor, as appropriate, or a cell having a CD40 and/or CD154 receptoron its surface, as appropriate. CD40 and/or CD154 protein binding toCD154 and/or CD40, respectively, can be determined using methods knownin the art including Biacore® screening, confocal immunofluorescentmicroscopy, immunoprecipitation, gel chromatography, determination ofinhibition of binding of antibodies that bind specifically to the CD40and/or CD154 binding domain of CD40 and/or CD154, as appropriate, ELISAusing a CD40 and/or CD154 labeled with a detectable tag such as anenzyme or chemiluminescent tag or yeast-2 hybrid technology.

The present invention also includes mimetopes of canine interleukin-4,canine or feline Flt-3 ligand, canine or feline CD40, canine or felineCD154, canine interleukin-5, canine interleukin-13, feline interferonalpha, or feline GM-CSF proteins of the present invention. As usedherein, a mimetope of an immunoregulatory protein of the presentinvention refers to any compound that is able to mimic the activity ofsuch a canine interleukin-4, canine or feline Flt-3 ligand, canine orfeline CD40, canine or feline CD1 54, canine interleukin-5, canineinterleukin-13, feline interferon alpha, or feline GM-CSF protein,respectively, often because the mimetope has a structure that mimics theparticular protein. Mimetopes can be, but are not limited to: peptidesthat have been modified to decrease their susceptibility to degradationsuch as all-D retro peptides; anti-idiotypic and/or catalyticantibodies, or fragments thereof; non-proteinaceous immunogenic portionsof an isolated protein (e.g., carbohydrate structures); and/or syntheticor natural organic molecules, including nucleic acids. Such mimetopescan be designed using computer-generated structures of proteins of thepresent invention. Mimetopes can also be obtained by generating randomsamples of molecules, such as oligonucleotides, peptides or otherorganic molecules, and screening such samples by affinity chromatographytechniques using the corresponding binding partner.

One embodiment of an immunoregulatory protein of the present inventionis a fusion portion that includes either a canine interleukin-4, canineor feline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF protein-containing domain, each attached to one or morefusion segments. Suitable fusion segments for use with the presentinvention include, but are not limited to, segments that can: link twoor more immunoregulatory proteins of the present invention, to formmultimeric forms of an immunoregulatory protein of the presentinvention; enhance a protein's stability; act as an immunopotentiator toenhance an immune response against an canine interleukin-4, canine orfeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF protein; and/or assist in purification of an canineinterleukin-4, canine or feline Flt-3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF protein (e.g., by affinitychromatography). A suitable fusion segment can be a domain of any sizethat has the desired function (e.g., imparts increased stability,imparts increased immunogenicity to a protein, and/or simplifiespurification of a protein). Fusion segments can be joined to aminoand/or carboxyl termini of the IL-4-containing domain, or the Flt-3ligand-containing domain, or the CD40-containing domain, or theCD154-containing domain, or the IL-5-containing domain, or theIL-3-containing domain, or the IFNα-containing domain, orGM-CSF-containing domain, of a protein and can be susceptible tocleavage in order to enable straight-forward recovery of either canineinterleukin-4, canine or feline Flt-3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF protein, respectively. Fusionproteins are preferably produced by culturing a recombinant celltransformed with a fusion nucleic acid molecule that encodes a proteinincluding the fusion segment attached to either the carboxyl and/oramino terminal end of an canine interleukin-4-, canine or feline Flt-3ligand-, canine or feline CD40-, canine or feline CD154-, canineinterleukin-5-, canine interleukin-13-, feline interferon alpha-, orfeline GM-CSF-containing domain. Preferred fusion segments include ametal binding domain (e.g., a poly-histidine segment); andimmunoglobulin binding domain (e.g., Protein A; Protein G; T cell; Bcell; Fc receptor or complement protein antibody-binding domains); asugar binding domain (e.g., a maltose binding domain); and/or a “tag”domain (e.g., at least a portion of -galactosidase, a strep tag peptide,a T7 tag peptide, a Flag™ peptide, or other domains that can be purifiedusing compounds that bind to the domain, such as monoclonal antibodies).More preferred fusion segments include metal binding domains, such as apoly-histidine segment; a maltose binding domain; a strep tag peptide,such as that available from Biometra in Tampa, Fla.; and an S10 peptide.

A suitable fusion segment that links one IL-4 protein to another IL-4protein, or one Flt-3 ligand protein to another Flt-3 ligand protein, orone CD40 protein to another CD40 protein, or one CD154 protein toanother CD154 protein, or one IL-5 protein to another IL-5 protein toanother IL-5 protein, or one IL-13 protein to another IL-13 protein, orone IFNα protein to another IFNα protein, or one GM-CSF protein toanother GM-CSF protein, includes any amino acid sequence that enablessuch proteins to be linked while maintaining the biological function ofeither the canine interleukin-4, canine or feline Flt-3 ligand, canineor feline CD40, canine or feline CD154, canine interleukin-5, canineinterleukin-13, feline interferon alpha, or feline GM-CSF, proteins,respectively. Selection of a suitable linker is dependent upon how manyproteins are to be linked to form one multimeric molecule and from whereon either the canine interleukin-4, canine or feline Flt-3 ligand,canine or feline CD40, canine or feline CD154, canine interleukin-5,canine interleukin-13, feline interferon alpha, or feline GM-CSFmolecule the linker extends. Preferably, a linker fusion segment of thepresent invention comprises a peptide of from about 6 amino acidresidues to about 40 residues, more preferably from about 6 residues toabout 30 residues in length.

In another embodiment, an canine interleukin-4, canine or feline Flt-3ligand, canine or feline CD40, canine or feline CD154, canineinterleukin-5,canine interleukin-13, feline interferon alpha, or felineGM-CSF protein of the present invention also includes at least oneadditional protein segment that is capable of targeting either canineinterleukin-4, canine or feline Flt-3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF protein, respectively, to adesired cell or receptive molecule. Such a multivalent targeting proteincan be produced by culturing a cell transformed with a nucleic acidmolecule comprising two or more nucleic acid domains joined together insuch a manner that the resulting nucleic acid molecule is expressed as amultivalent targeting protein containing a canine interleukin-4, canineor feline Flt-3 ligand, canine or feline CD40, canine or feline CD1 54,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF protein or portion thereof and/or at least one targetingcompound capable of delivering the canine interleukin-4, canine offeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13feline interferon alpha, orfeline GM-CSF protein, respectively, to a desired site in an animal.

Examples of multivalent targeting proteins include, but are not limitedto, a canine interleukin-4, canine or feline Flt-3 ligand, canine orfeline CD40, canine or feline CD154, canine interleukin-5, canineinterleukin-13, feline interferon alpha, or feline GM-CSF protein of thepresent invention attached to one or more compounds that can bind to areceptive molecule on the surface of a cell located in an area of ananimal where regulation of an immune response is desired. One of skillin the art can select appropriate targeting fusion segments dependingupon the cell or receptive molecule being targeted.

Another example of a multivalent protein of the present inventionincludes, but is not limited to, a canine interleukin-4, canine orfeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF protein of the present invention attached to one or moreproteins that are potentially antigenic in mammals. Thus, immunogenicityof the potentially antigenic protein could be enhanced by administeringto a mammal together with an immunoregulatory protein of the presentinvention.

A naturally-occurring variant of a canine interleukin-4, canine orfeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF protein of the present invention is preferably isolatedfrom (including isolation of the natural protein or production of theprotein by recombinant or synthetic techniques) from mammals, includingbut not limited to dogs (i.e., canids), cats (i.e., felids), horses(i.e., equids), humans, cattle, chinchillas, ferrets, goats, mice,minks, rabbits, raccoons, rats, sheep, squirrels, swine, chickens,ostriches, quail and/or turkeys as well as other furry animals, pets,zoo animals, work animals and/or food animals. Particularly preferredanimals from which to isolate canine interleukin-4, canine or felineFlt-3 ligand, canine or feline CD40, canine or feline CD154, canineinterleukin-5, canine interleukin-13, feline interferon alpha, or felineGM-CSF proteins are dogs, cats, horses and/or humans.

A preferred isolated protein of the present invention is a proteinencoded by at least one of the following nucleic acidmolecules:nCaIL-4₅₄₉, nCaIL-4₃₉₆, nCaIL-4₃₂₄, nCaFlt3L₁₀₁₃, nCaFlt3L₈₈₂,nCaFlt3L₈₀₄, nCaFlt3L₈₂₈, nCaFlt3L₉₈₅, nCaFlt3L₁₀₁₉, nCaFlt3L₉₃,nCaFlt3L₇₅₀, nFeFlt3L₃₉₅, nFeFlt3L₇₉₃, nFeFlt3L₉₄₂, nFeFlt3L₈₇₃,nFeFlt3L₇₉₅, nCaCD40₃₂₁, nCaCD40₁₄₂₅, nCaCD40₈₂₂, nCaCD40₇₆₅,nFeCD40₃₃₆, nCaCD154₃₉₀, nCaCD1541₈₇₈, nCaCD154₇₈₀, nCaCD154₆₃₃,nFeCD154₈₈₅, nFeCD154₇₈₀, nFeCD154₆₃₃, nCaIL-5₆₁₀, nCaIL-5₄₀₂,nCaIL-5₃₄₅, nCaIL-13₁₆₆, nCaIL-13₂₇₂, nCaIL-13₂₇₈, nCaIL-13₁₃₀₂,nCaIL-13₃₉₃, nCaIL-13₃₃₃, nCaIL-13₁₂₆₉, nCaIL-13₃₉₀, nCaIL-13₃₃₀,nFeIFNα_(567a), nFeIFNα_(567b), nFeIFNα_(567c), nFeIFNα_(498a),nFeIFNα_(498b), nFeIFNα_(498c), nFeIFNα_(582d), nFeIFNα_(513d),nFeIFNα_(567e), nFeIFNα_(498e), nFeGMCSF₄₄₄, nFeGMCSF₄₃₂, nFeGMCSF₃₈,and/or allelic variants of any of these nucleic acid molecules. Alsopreferred is an isolated protein that is encoded by a nucleic acidmolecule the having nucleic acid sequence SEQ ID NO:1, SEQ ID NO:4, SEQID NO:19, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:22, SEQ ID NO:25, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:88, SEQ IDNO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:99, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, SEQID NO:113, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ IDNO:167, and SEQ ID NO:170; and/or an allelic variant of such a nucleicacid molecule.

Translation of SEQ ID NO:1, the coding strand of nCaIL-4₅₄₉, yields aprotein of about 132 amino acids, denoted herein as PCaIL-4₁₃₂, theamino acid sequence of which is presented in SEQ ID NO:2, assuming anopen reading frame having an initiation codon spanning from nucleotide43 through nucleotide 45 of SEQ ID NO:1 and a stop codon spanning fromnucleotide 439 through nucleotide 441 of SEQ ID NO:1.

Translation of SEQ ID NO:6, the coding strand of nCaFlt3L₁₀₁₃, yields aprotein of about 294 amino acids, denoted herein as PCaFlt3L₂₉₄, theamino acid sequence of which is presented in SEQ ID NO:7, assuming anopen reading frame having an initiation codon spanning from nucleotide35 through nucleotide 37 of SEQ ID NO:6 and a stop codon spanning fromnucleotide 917 through nucleotide 919 of SEQ ID NO:6.

Translation of SEQ ID NO:43, the coding strand for nFeFlt3L₉₄₂, yields aprotein of about 291 amino acids, denoted herein as PFeFlt3L₂₉₁, theamino acid sequence of which is presented in SEQ ID NO:44, assuming anopen reading frame having an initiation codon spanning from nucleotide31 through nucleotide 33 of SEQ ID NO:43 and a stop codon spanning fromnucleotide 904 through nucleotide 906 of SEQ ID NO:43.

Translation of SEQ ID NO:52, the coding strand for nCaCD40₁₄₂₅, yields aprotein of about 274 amino acids, denoted herein as PCaCD40₂₇₄, theamino acid sequence of which is presented in SEQ ID NO:53, assuming anopen reading frame having an initiation codon spanning from nucleotide196 through nucleotide 198 of SEQ ID NO:52 and a stop codon spanningfrom about nucleotide 1018 through nucleotide 1020 of SEQ ID NO:52.

Translation of SEQ ID NO:60, the coding strand for nFeCD40₃₃₆, yields aprotein of about 112 amino acids, denoted herein as PFeCD40₁₁₂, theamino acid sequence of which is presented in SEQ ID NO:61, assuming anopen reading frame having an initiation codon spanning from nucleotide 1through nucleotide 3 of SEQ ID NO:60.

Translation of SEQ ID NO:64, the coding strand for nCaCD154₁₈₇₈, yieldsa protein of about 260 amino acids, denoted herein as PCaCD154₂₆₀, theamino acid sequence of which is presented in SEQ ID NO:65, assuming anopen reading frame having an initiation codon spanning from nucleotide284 through nucleotide 286 of SEQ ID NO:64 and a stop codon spanningfrom nucleotide 1064 through nucleotide 1066 of SEQ ID NO:64.

Translation of SEQ ID NO:72, the coding strand for nFeCD154₈₈₅, yields aprotein of about 260 amino acids, denoted herein as PFeCD154₂₆₀, theamino acid sequence of which is presented in SEQ ID NO:73, assuming anopen reading frame having an initiation codon spanning from nucleotide29 through nucleotide 31 of SEQ ID NO:72, and a stop codon spanning fromnucleotide 809 through nucleotide 811 of SEQ ID NO:72.

Translation of SEQ ID NO:80, the coding strand for nCaIL-5₆₁₀, yields aprotein of about 134 amino acids, denoted herein as PCaIL-5₁₃₄, theamino acid sequence of which is presented in SEQ ID NO:81, assuming anopen reading frame having an initiation codon spanning from nucleotide29 through nucleotide 31 of SEQ ID NO:80, and a stop codon spanning fromnucleotide 431 through nucleotide 433 of SEQ ID NO:80.

Translation of SEQ ID NO:91, the coding stand for nCaIL-13₁₃₀₂, yields aprotein of about 131 amino acids, denoted herein as PCaIL-13₁₃₁, theamino acid sequence of which is presented in SEQ ID NO:92, assuming anopen reading frame having an initiation codon spanning from nucleotide52 through nucleotide 54 of SEQ ID NO:91 and a stop codon spanning fromnucleotide 445 through nucleotide 447 of SEQ ID NO:91.

Translation of SEQ ID NO:107, the coding strand for nFeIFNα_(567a),yields a protein of about 189 amino acids, denoted herein asPFeIFNα_(189a), the amino acid sequence of which is presented in SEQ IDNO:108, assuming an open reading frame having an initiation codonspanning from nucleotide 1 through nucleotide 3 and a last codon priorto a stop codon spanning from nucleotide 565 through nucleotide 567 ofSEQ ID NO:107.

Translation of SEQ ID NO:110, the coding strand for nFeIFNα_(567b),yields a protein of about 189 amino acids, denoted herein asPFeIFNα_(189b), the amino acids sequence of which is presented in SEQ IDNO:111, assuming an open reading frame having an initiation codonspanning from nucleotide 1 through nucleotide 3 and a last codon priorto a stop codon spanning from nucleotide 565 through nucleotide 567 ofSEQ ID NO:110.

Translation of SEQ ID NO:155, the coding strand for nFeIFNα_(567c),yields a protein of about 189 amino acids, denoted herein asPFeIFNα_(189c), the amino acid sequence of which is presented in SEQ IDNO:156, assuming an open reading frame having an initiation codonspanning from nucleotide 1 through nucleotide 3 and a last codon priorto a stop codon spanning from nucleotide 565 through nucleotide 567 ofSEQ ID NO:155.

Translation of SEQ ID NO:161, the coding strand for nFeIFNα_(582d),yields a protein of about 194 amino acids, denoted herein asPFeIFNα_(194d), the amino acid sequence of which is presented in SEQ IDNO:162, assuming an open reading frame having an initiation codonspanning from nucleotide 1 through nucleotide 3 and a last codon priorto a stop codon spanning from nucleotide 565 through nucleotide 567 ofSEQ ID NO:161.

Translation of SEQ ID NO:167, the coding strand for nFeIFNα_(567e),yields a protein of about 189 amino acids, denoted herein asPFeIFNα_(189e), the amino acid sequence of which is presented in SEQ IDNO:168, assuming an open reading frame having an initiation codonspanning from nucleotide 1 through nucleotide 3 and a last codon priorto a stop codon spanning from nucleotide 565 through nucleotide 567 ofSEQ ID NO:167.

Translation of SEQ ID NO:119, the coding strand for nFeGMCSF₄₄₄, yieldsa protein of about 144 amino acids, denoted herein as PFeGMCSF₁₄₄, theamino acid sequence of which is presented in SEQ ID NO:120, assuming anopen reading frame having an initiation codon spanning from nucleotide10 through nucleotide 12 of SEQ ID NO:119 and a stop codon spanning fromnucleotide 442 through nucleotide 444 of SEQ ID NO:119.

Preferred IL-4 proteins of the present invention include proteins thatare at least about 85%, preferably at least about 90%, and even morepreferably at least about 95% identical to PCaIL-4₁₃₂, PCaIL-4₁₀₈, orfragments thereof. Preferred Flt-3 ligand proteins of the presentinvention include proteins that are at least about 75%, even morepreferably at least about 80%, even more preferably at least about 85%,even more preferably at least about 90%, and even more preferably atleast about 95% identical to PCaFlt3L₂₉₄, PCaFlt3L₂₆₈, PCaFlt3L₂₇₆,PCaFlt3L₂₅₀, PCaFlt3L₃₁, and/or fragments thereof. Additional preferredFlt-3 ligand proteins of the present invention includes proteins thatare at least about 75%, even more preferably at least about 80%, evenmore preferably at least about 85%, even more preferably at least about90%, and even more preferably at least about 95% identical toPFeFlt3L₂₉₁, PFeFlt3L₂₆₅ and/or fragments thereof. Preferred CD40proteins of the present invention includes proteins that are at leastabout 70%, preferably at least about 75%, even more preferably at leastabout 80%, even more preferably at least about 85%, even more preferablyat least about 90%, and even more preferably at least about 95%identical to PCaCD40₂₇₄, PCaCD40₂₅₅ and/or fragments thereof Additionalpreferred CD40 proteins of the present invention includes proteins thatare at least about 60%, at least about 65%, preferably at least about70%, preferably at least about 75%, even more preferably at least about80%, even more preferably at least about 85%, even more preferably atleast about 90%, and even more preferably at least about 95% identicalto PFeCD40₁₁₂ and/or fragments thereof. Preferred CD154 proteins of thepresent invention includes proteins that are at least about 80%identical, preferably at least about 85% identical, even more preferablyat least about 90%, and even more preferably at least about 95%identical to PCaCD154₂₆₀, PCaCD154₂₁₁ and/or fragments thereof.Additional preferred CD154 proteins of the present invention includesproteins that are at least about 85% identical, even more preferably atleast about 90%, and even more preferably at least about 95% identicalto PFeCD154₂₆₀, PFeCD154₂₁₁ and/or fragments thereof. Preferred IL-5proteins of the present invention includes proteins that are at leastabout 85% identical, even more preferably at least about 90%, and evenmore preferably at least about 95% identical to PCaIL-5₁₃₄, PCaIL-5₁₁₅and/or fragments thereof Preferred IL-13 proteins of the presentinvention includes proteins that are at least about 70% identical,preferably at least about 75% identical, more preferably at least about80% identical, more preferably at least about 85% identical, even morepreferably at least about 90%, and even more preferably at least about95% identical to PCaIL-13₁₃₁, PCaIL-13₁₁₁, PCaEL-13₁₃₀, PCaIL-13₁₁₀,and/or fragments thereof. Preferred IFNα proteins of the presentinvention include PFeIFNα_(189a), PFeIFNα_(189b), PFeIFNα_(189c),PFeIFNα_(166a), PFeIFNα_(166c), PFeIFNα_(194d), PFeIFNα_(171d),PFeIFNα_(189e), PFeIFNα_(166e), and/or PFeIFNα_(166b). Preferred GM-CSFproteins of the present invention include PFeGMCSF₁₄₄, and/orPFeGMCSF₁₂₇.

More preferred are IL-4 proteins comprising PCaIL-4₁₃₂, PCaIL-4₁₀₈,and/or proteins encoded by allelic variants of a nucleic acid moleculeencoding proteins PCaIL-4₁₃₂ and/or PCaEL-4₁₀₈. More preferred are Flt-3ligand proteins comprising PCaFlt3L₂₉₄, PCaFlt3L₂₆₈, PCaFlt3L₂₇₆,PCaFlt3L₂₅₀, PCaFlt3L₃₁, PFeFlt3L₂₉₁, PFeFlt3L₂₆₅ and/or proteinsencoded by allelic variants of a nucleic acid molecule encoding proteinsPCaFlt3L₂₉₄, PCaFlt3L₂₆₈, PCaFlt3L₂₇₆, PCaFlt3L₂₅₀ PCaFlt3L₃₁,PFeFlt3L₂₉₁, and/or PFeFlt3L₂₆₅. More preferred are CD40 proteinscomprising PCaCD40₂₇₄, PCaCD40₂₅₅, and/or PFeCD40₁₁₂ and/or proteinsencoded by allelic variants of a nucleic acid molecule encoding proteinsPCaCD40₂₇₄, PCaCD40₂₅₅, and/or PFeCD40₁₁₂. More preferred are CD154proteins comprising PCaCD154₂₆₀, PCaCD154₂₁₁, PFeCD154₂₆₀, PFeCD154₂₁₁and/or proteins encoded by allelic variants of a nucleic acid moleculeencoding one of proteins PCaCD154₂₆₀, PCaCD154₂₁₁ PFeCD154₂₆₀,PFeCD154₂₁₁. More preferred are IL-5 proteins comprising PCaEL-5₁₃₄,PCaIL-5₁₁₅ and/or proteins encoded by allelic variants of a nucleic acidmolecule encoding one of the proteins PCaIL-5₁₃₄ and/or PCaIL-5₁₁₅. Morepreferred are IL-13 proteins comprising PCaIL-13₁₃₁, PCaIL-13₁₁₁,PCaIL-13₁₃₀, PCaIL-13₁₁₀, and/or proteins encoded by allelic variants ofanucleic acid molecule encoding one of the proteins PCaIL-13₁₃₁,PCaIL-13₁₁₁, PCaIL-13₁₃₀, PCaIL-13₁₁₀.

Also preferred are IL-4 proteins of the present invention having aminoacid sequences that are at least about 85%, preferably at least about90%, and even more preferably at least about 95% identical to SEQ IDNO:2, SEQ ID NO:20 and/or fragments thereof Also preferred are Flt-3ligand proteins of the present invention having amino acid sequencesthat are at least about 75%, even more preferably at least about 80%,even more preferably at least about 85%, even more preferably at leastabout 90%, and even more preferably at least about 95% identical to SEQID NO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, and/or SEQ ID NO:34and/or fragments thereof. Additional preferred Flt-3 ligand proteins ofthe present invention includes proteins that are at least about 75%,even more preferably at least about 80%, even more preferably at leastabout 85%, even more preferably at least about 90%, and/or even morepreferably at least about 95% identical to SEQ ID NO:44, SEQ ID NO:49and/or fragments thereof. Preferred CD40 proteins of the presentinvention includes proteins that are at least about 70%, preferably atleast about 75%, even more preferably at least about 80%, even morepreferably at least about 85%, even more preferably at least about 90%,and/or even more preferably at least about 95% identical to SEQ IDNO:53, SEQ ID NO:58 and/or fragments thereof. Additional preferred CD40proteins of the present invention includes proteins that are at leastabout 60%, at least about 65%, preferably at least about 70%, preferablyat least about 75%, even more preferably at least about 80%, even morepreferably at least about 85%, even more preferably at least about 90%,and even more preferably at least about 95% identical to SEQ ID NO:61and/or fragments thereof. Preferred CD154 proteins of the presentinvention includes proteins that are at least about 80% identical,preferably at least about 85% identical, even more preferably at leastabout 90%, and even more preferably at least about 95% identical to SEQID NO:65, SEQ ID NO:70 and/or fragments thereof. Additional preferredCD154 proteins of the present invention includes proteins that are atleast about 85% identical, even more preferably at least about 90%, andeven more preferably at least about 95% identical to SEQ ID NO:73, SEQID NO:78 and/or fragments thereof. Preferred IL-5 proteins of thepresent invention includes proteins that are at least about 85%identical, even more preferably at least about 90%, and even morepreferably at least about 95% identical to SEQ ID NO:81, SEQ ID NO:86and/or fragments thereof. Preferred IL-13 proteins of the presentinvention includes proteins that are at least about 70% identical,preferably at least about 75% identical, more preferably at least about80% identical, more preferably at least about 85% identical, even morepreferably at least about 90%, and even more p preferably at least about95% identical to SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO:100, SEQ IDNO:105, and/or fragments thereof. Preferred IFNα proteins of the presentinvention include SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ IDNO:117, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQID NO:168, and SEQ ID NO:171. Preferred GM-CSF proteins of the presentinvention include SEQ ID NO:120, SEQ ID NO:125.

More preferred are IL-4 proteins comprising the amino acid sequence SEQID NO:2, SEQ ID NO:20; and/or L-4 proteins encoded by allelic variantsof nucleic acid molecules encoding IL-4 proteins having the amino acidsequence SEQ ID NO:2, SEQ ID NO:20. More preferred are Flt-3 ligandproteins comprising SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ IDNO:31, and/or SEQ ID NO:34, SEQ ID NO:44, SEQ ID NO:49 and/or proteinsencoded by allelic variants of a nucleic acid molecule encoding proteinsSEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:34, SEQID NO:44, and/or SEQ ID NO:49. More preferred are CD40 proteinscomprising SEQ ID NO:53, SEQ ID NO:58 SEQ ID NO:61 and/or proteinsencoded by allelic variants of a nucleic acid molecule encoding proteinsSEQ ID NO:53, SEQ ID NO:58 and/or SEQ ID NO:61. More preferred are CD154proteins comprising SEQ ID NO:65, SEQ ID NO:70 SEQ ID NO:73, SEQ IDNO:78 and/or proteins encoded by allelic variants of a nucleic acidmolecule encoding one of proteins SEQ ID NO:65, SEQ ID NO:70, SEQ IDNO:73, and/or SEQ ID NO:78. More preferred are IL-5 proteins comprisingSEQ ID NO:81, SEQ ID NO:86 and/or proteins encoded by allelic variantsof a nucleic acid molecule encoding one of the proteins SEQ ID NO:81,and/or SEQ ID NO:86. More preferred are IL-13 proteins comprising SEQ IDNO:92, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:105, and/or proteinsencoded by allelic variants of anucleic acid molecule encoding one ofthe proteins SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO:100, and/or SEQ IDNO:105.

Percent identities between amino acid or nucleic acid sequences can bedetermined using standard methods known to those of skill in the art. Itis known in the art that methods to determine the percentage identityand the number of gaps are substantially similar when different methodsfor determining sequence similarity are used and when the degree ofsimilarity is greater than 30% amino acid identity, as described inJohnson et al., J. Mol. Biol., vol. 233, pages 716-738, 1993, and Fenget al., J. Mol. Evol., vol. 21, pages 112-125, 1985, which areincorporated by reference herein in their entirety. Preferred methods todetermine percentage identities between amino acid sequences and betweennucleic acid sequences include comparisons using various computerprograms such as GCG™ program (available from Genetics Computer Group,Madison, Wis.), DNAsis™ program (available from Hitachi Software, SanBruno, Calif.) or the MacVector™ program (available from the EastmanKodak Company, New Haven, Conn.). Preferred settings for sequencecomparisons using the DNAsis™ computer program or the GAPGCG™ programare disclosed herein in the Examples section.

Additional preferred L-4 proteins of the present invention includeproteins encoded by nucleic acid molecules comprising at least a portionof nCaIL-4₅₄₉, nCaIL-4₃₉₆, and/or nCaIL-4₃₂₄, as well as IL-4 proteinsencoded by allelic variants of such nucleic acid molecules. Additionalpreferred Flt-3 ligand proteins of the present invention includeproteins encoded by nucleic acid molecules comprising at least a portionof nCaFlt3L₁₀₁₃, nCaFlt3L₈₈₂, nCaFlt3L₈₀₄, nCaFlt3L₈₂₈, nCaFlt3L₉₈₅,CaFlt3L₁₀₉, nCaFlt3L₉₃, nCaFlt3L₇₅₀, nFeFlt3L₃₉₅, nFeFlt3L₇₉₃,nFeFlt3L₉₄₂, nFeFlt3L₈₇₃, and/or nFeFlt3L₇₉₅ as well as Flt-3 ligandproteins encoded by allelic variants of such nucleic acid molecules.Additional preferred CD40 proteins of the present invention includeproteins encoded by nucleic acid molecules encoding at least a proteinof nCaCD40₃₂₁, nCaCD40₁₄₂₅, nCaCD40₈₂₂, nCaCD40₇₆₅, and/or nFeCD40₃₃₆ aswell as CD40 proteins encoded by allelic variants of such nucleic acidmolecules. Additional preferred CD154 proteins of the present inventioninclude proteins encoded by nucleic acid molecules encoding at least aportion of nCaCD154₃₉₀, nCaCD1541₈₇₈, nCaCD154₇₈₀, nCaCD154₆₃₃,nFeCD154₈₈₅, nFeCD154₇₈₀, and/or nFeCD154₆₃₃ as well as CD154 proteinsencoded by allelic variants of such nucleic acid molecules. Additionalpreferred IL-5 proteins of the present invention include proteinsencoded by nucleic acid molecules encoding at least a portion ofnCaIL-5₆₁₀, nCaIL-5₄₀₂, and/or nCaIL-5₃₄₅ as well as IL-5 proteinsencoded by allelic variants of such nucleic acid molecules. Additionalpreferred IL-13 proteins of the present invention include proteinsencoded by nucleic acid molecules encoding at least a portion ofnCaIL-5₆₁₀, nCaIL-5₄₀₂, and/or nCaIL-5₃₄₅ as well as IL-13 proteinsencoded by allelic variants of such nucleic acid molecules.

Also preferred are IL-4 proteins encoded by nucleic acid moleculeshaving nucleic acid sequences comprising at least a portion of SEQ IDNO:1, SEQ ID NO:4, and/or SEQ ID NO:19, as well as allelic variants ofthese nucleic acid molecules. Also preferred are Flt-3 ligand proteinsencoded by nucleic acid molecules having nucleic acid sequencescomprising at least a portion of SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:22,SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46, and/or SEQ IDNO:48, as well as allelic variants of these nucleic acid molecules. Alsopreferred are CD40 proteins encoded by nucleic acid molecules havingnucleic acid sequences comprising at least a portion of SEQ ID NO:51,SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:57, and/or SEQ ID NO:60, as wellas allelic variants of these nucleic acid molecules. Also preferred areCD154 proteins encoded by nucleic acid molecules having nucleic acidsequences comprising at least a portion of SEQ ID NO:63, SEQ ID NO:64,SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:75, and/or SEQ IDNO:77, as well as allelic variants of these nucleic acid molecules. Alsopreferred are IL-5 proteins encoded by nucleic acid molecules havingnucleic acid sequences comprising at least a portion of SEQ ID NO:80,SEQ ID NO:83, and/or SEQ ID NO:85, as well as allelic variants of thesenucleic acid molecules. Also preferred are EL-13 proteins encoded bynucleic acid molecules having nucleic acid sequences comprising at leasta portion of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQID NO:94, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, and/or SEQ IDNO:104, as well as allelic variants of these nucleic acid molecules.

Another embodiment of the present invention is a canine interleukin-4,canine or feline Flt-3 ligand, canine or feline CD40, canine or felineCD154, canine interleukin-5, canine interleukin-13, feline interferonalpha, or feline GM-CSF nucleic acid molecule that includes one or moreregulatory regions, full-length or partial coding regions, orcombinations thereof The minimal size of a nucleic acid molecule of thepresent invention is a size sufficient to allow the formation of astable hybrid (i.e., hybridization under stringent hybridizationconditions) with the complementary sequence of another nucleic acidmolecule. As such, the minimal size of a canine interleukin-4, canine orfeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF nucleic acid molecule of the present invention is fromabout 12 to about 18 nucleotides in length.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subjected to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated canine interleukin-4, canine orfeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha, orfeline GM-CSF nucleic acid molecule of the present invention can beisolated from its natural source or produced using recombinant DNAtechnology (e.g., polymerase chain reaction (PCR) amplification orcloning) or chemical synthesis. Isolated canine interleukin-4, canine orfeline Flt-3 ligand, canine or feline CD40, canine or feline CD154,canine interleukin-5, canine interleukin-13, feline interferon alpha,and/or feline GM-CSF, nucleic acid molecules can include, for example,natural allelic variants and/or nucleic acid molecules modified bynucleotide insertions, deletions, substitutions, and/or inversions in amanner such that the modifications do not substantially interfere withthe nucleic acid molecule's ability to encode an canine interleukin-4,canine or feline Flt-3 ligand, canine or feline CD40, canine or felineCD154, canine interleukin-5, canine interleukin-13, feline interferonalpha, and/or feline GM-CSF protein of the present invention.

A canine interleukin-4, canine or feline Flt-3 ligand, canine or felineCD40, canine or feline CD154, canine interleukin-5, canineinterleukin-13, feline interferon alpha, and/or feline GM-CSF ligandnucleic acid molecule homolog can be produced using a number of methodsknown to those skilled in the art, see, for example, Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress; Sambrook et al., ibid., is incorporated by reference herein inits entirety. For example, nucleic acid molecules can be modified usinga variety of techniques including, but not limited to, classicmutagenesis and recombinant DNA techniques such as site-directedmutagenesis, chemical treatment, restriction enzyme cleavage, ligationof nucleic acid fragments, PCR amplification, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules, and combinations thereof. Nucleicacid molecule homologs can be selected by hybridization with either acanine interleukin-4, canine or feline Flt-3 ligand, canine or felineCD40, canine or feline CD154, canine interleukin-5, canineinterleukin-13, feline interferon alpha, or feline GM-CSF nucleic acidmolecule or by screening the function of a protein encoded by thenucleic acid molecule (e.g., ability to elicit an immune responseagainst at least one epitope of a canine interleukin-4, canine or felineFlt-3 ligand, canine or feline CD40, canine or feline CD154, canineinterleukin-5, canine interleukin-13, feline interferon alpha, or felineGM-CSF protein, respectively).

An isolated nucleic acid molecule of the present invention can include anucleic acid sequence that encodes at least one canine interleukin-4,canine or feline Flt-3 ligand, canine or feline CD40, canine or felineCD154, canine interleukin-5, canine interleukin-13, feline interferonalpha, or feline GM-CSF protein of the present invention, examples ofsuch proteins being disclosed herein. Although the phrase “nucleic acidmolecule” primarily refers to the physical nucleic acid molecule and thephrase “nucleic acid sequence” primarily refers to the sequence ofnucleotides on the nucleic acid molecule, the two phrases can be usedinterchangeably, especially with respect to a nucleic acid molecule, ora nucleic acid sequence, being capable of encoding a canineinterleukin-4, canine or feline Flt3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF ligand protein.

A preferred nucleic acid molecule of the present invention, whenadministered to an animal, is capable of regulating an immune responsein an animal. As will be disclosed in more detail below, such a nucleicacid molecule can be, or encode, an antisense RNA, a molecule capable oftriple helix formation, a ribozyme, or other nucleic acid-based drugcompound. In additional embodiments, a nucleic acid molecule of thepresent invention can encode an immunoregulatory protein (e.g., acell-bound or soluble protein of the present invention), the nucleicacid molecule being delivered to the animal, for example, by directinjection (i.e., as a genetic vaccine) or in a vehicle such as arecombinant virus vaccine or a recombinant cell vaccine.

One embodiment of the present invention is an IL-4 nucleic acid moleculecomprising all or part (i.e., a fragment of the IL-4 nucleic acidmolecule) of nucleic acid molecules nCaIL-4₅₄₉, nCaIL-4₃₉₆, and/ornCaIL-4₃₂₄, or allelic variants of these nucleic acid molecules. Oneembodiment of the present invention is a Flt-3 ligand nucleic acidmolecule comprising all or part (i.e., a fragment of the Flt-3 ligandnucleic acid molecule) of nucleic acid molecules nCaFlt3L₁₀₁₃,nCaFlt3L₈₈₂, nCaFlt3L₈₀₄, nCaFlt3L₈₂₈, nCaFlt3L₉₈₅, nCaFlt3L₁₀₁₉,nCaFlt3L₉₃, nCaFlt3L₇₅₀, nFeFlt3L₃₉₅, nFeFlt3L₇₉₃, nFeFlt3L₉₄₂,nFeFlt3L₈₇₃, and/or nFeFlt3L₇₉₅, and/or allelic variants of thesenucleic acid molecules. One embodiment of the present invention is aCD40 nucleic acid molecule comprising all or part (i.e. a fragment ofthe CD40 nucleic acid molecule) of nucleic acid molecules nCaCD40₃₂₁,nCaCD40₁₄₂₅, nCaCD40₈₂₂, nCaCD40₇₆₅, and/or nFeCD40₃₃₆ and/or allelicvariants of these nucleic acid molecules. One embodiment of the presentinvention is a CCD154 nucleic acid molecule comprising all or part ofnucleic acid molecules nCaCD154₃₉₀, nCaCD1541₈₇₈, nCaCD154₇₈₀,nCaCD154₆₃₃, nFeCD154₈₈₅, nFeCD154₇₈₀, and/or nFeCD154₆₃₃, and/orallelic variants of these nucleic acid molecules. One embodiment of thepresent invention is an IL-5 nucleic acid molecule comprising all orpart of nucleic acid molecules nCaIL-5₆₁₀, nCaIL-5₄₀₂, and/ornCaIL-5₃₄₅, and/or allelic variants of these nucleic acid molecules. Oneembodiment of the present invention is an IL-13 nucleic acid moleculecomprising all or part of nucleic acid molecules nCaIL-13₁₆₆,nCaIL-13₂₇₂, nCaIL-13₂₇₈, nCaIL-13₁₃₀₂, nCaIL-13₃₉₃, nCaIL-13₃₃₃,nCaIL-13₁₂₆₉, nCaIL-13₃₉₀, and/or nCaIL-13₃₃₀, and/or allelic variantsof these nucleic acid molecules. Another preferred nucleic acid moleculeof the present invention includes at least a portion of (i.e., afragment of the nucleic acid molecule) nucleic acid sequence SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ IDNO:104, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ IDNO:124, SEQ ID NO:126, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:158, SEQID NO:160, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:166,SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:170, and/or SEQ ID NO:172, aswell as allelic variants of nucleic acid molecules having these nucleicacid sequences. Such nucleic acid molecules can include nucleotides inaddition to those included in the SEQ ID NOs, such as, but not limitedto, a full-length gene, a full-length coding region, a nucleic acidmolecule encoding a fusion protein, and/or a nucleic acid moleculeencoding a multivalent therapeutic compound.

One embodiment of an isolated nucleic acid molecule of the presentinvention is a nucleic acid molecule that can be any of the following:(a) an isolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:19, and/or SEQ ID NO:21 and/or a homologthereof, wherein said homolog has an at least 50 contiguous nucleotideregion identical in sequence to a 50 contiguous nucleotide region of anucleic acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, and/or SEQ IDNO:21; (b) an isolated nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQID NO:27, SEQ ED NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:36, and/or SEQ ID NO:37, and/or a homologthereof, wherein said homolog has an at least 40 contiguous nucleotideregion identical in sequence to a 40 contiguous nucleotide region of anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35,SEQ ID NO:36, and/or SEQ ID NO:37; (c) an isolated nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, and/or SEQ ID NO:50, and/or a homologthereof, wherein said homolog has an at least 30 contiguous nucleotideregion identical in sequence to a 30 contiguous nucleotide region of anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and/or SEQ ID NO:50;(d) an isolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and/or SEQ ID NO:59,and/or a homolog thereof, wherein said homolog has an at least 40contiguous nucleotide region identical in sequence to a 40 contiguousnucleotide region of a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and/orSEQ ID NO:59; (e) an isolated nucleic acid molecule comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO:60 and/orSEQ ID NO:62, and/or a homolog thereof, wherein said homolog has an atleast 30 contiguous nucleotide region identical in sequence to a 30contiguous nucleotide region of a nucleic acid molecule having a nucleicacid sequence selected from the group consisting of SEQ ID NO:60 and/orSEQ ID NO:62; (f) an isolated nucleic acid molecule comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69 and/or SEQID NO:71, and/or a homolog thereof, wherein said homolog has an at least45 contiguous nucleotide region identical in sequence to a 45 contiguousnucleotide region of a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO:63, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and/orSEQ ID NO:71; (g) an isolated nucleic acid molecule comprising a nucleicacid sequence selected from the group consisting of SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and/or SEQ ID NO:79,and/or a homolog thereof, wherein said homolog has an at least 35contiguous nucleotide region identical in sequence to a 35 contiguousnucleotide region of a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, and/or SEQ ID NO:79;(h) an isolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, and/or SEQ ID NO:87, and/or a homologthereof, wherein said homolog has an at least 45 contiguous nucleotideregion identical in sequence to a 45 contiguous nucleotide region of anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, SEQ ID NO:85, and/or SEQ ID NO:87; (i) an isolated nucleic acidmolecule comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91,SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98,SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ IDNO:104, and/or SEQ ID NO:106, and/or a homolog thereof, wherein saidhomolog has an at least 15 contiguous nucleotide region identical insequence to a 15 contiguous nucleotide region of a nucleic acid moleculehaving a nucleic acid sequence selected from the group consisting of SEQID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, and/or SEQ IDNO:106; (j) an isolated nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO:107, SEQ IDNO:109, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:158,SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ IDNO:166, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:170 and/or SEQ IDNO:172; and/or (k) an isolated nucleic acid molecule having a nucleicacid sequence selected from the group consisting of SEQ ID NO:119, SEQID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, and/or SEQ IDNO:126. The phrase, a homolog having an at least “x” contiguousnucleotide region identical in sequence to an “x” contiguous nucleotideregion of a nucleic acid molecule selected from the group consisting ofSEQ ID NO:“y”, refers to an “x”-nucleotide in length nucleic acidmolecule that is identical in sequence to an “x”-nucleotide portion ofSEQ ID NO:“y”, as well as to nucleic acid molecules that are longer inlength than “x”. The additional length may be in the form of nucleotidesthat extend from either the 5′ or the 3′ end(s) of the contiguousidentical “x”-nucleotide portion. The 5′ and/or 3′ extensions caninclude one or more extensions that have no identity to animmunoregulatory molecule of the present invention, as well asextensions that show similarity or identity to cited nucleic acidssequences or proteins thereof.

In another embodiment, an isolated nucleic acid molecule of the presentinvention can be any of the following: (a) a nucleic acid moleculehaving a nucleic acid sequence encoding an IL-4 protein selected fromthe group consisting of (i) a protein having an amino acid sequence thatis at least about 85 percent identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:2 and/or SEQ ID NO:20and/or (ii) a protein comprising a fragment of at least 20 amino acidsof an amino acid sequence selected from the group consisting of SEQ IDNO:2 and/or SEQ ID NO:20; (b) a nucleic acid molecule having a nucleicacid sequence encoding a Flt-3 ligand protein selected from the groupconsisting of (i) a protein having an amino acid sequence that is atleast about 75 percent identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ IDNO:31, and/or SEQ ID NO:34, and/or (ii) a protein comprising a fragmentof at least 25 amino acids of an amino acid sequence selected from thegroup consisting of SEQ ID NO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, and/or SEQ ID NO:34; (c) a nucleic acid molecule having a nucleicacid sequence encoding a Flt-3 ligand protein selected from the groupconsisting of (i) a protein having an amino acid sequence that is atleast about 75 percent identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:44 and/or SEQ ID NO:49 and/or (ii) aprotein comprising a fragment of at least 25 amino acids of an aminoacid sequence selected from the group consisting of SEQ ID NO:44 and/orSEQ ID NO:49; (d) a nucleic acid molecule having a nucleic acid sequenceencoding a CD40 protein selected from the group consisting of (i) aprotein having an amino acid sequence that is at least about 70 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:53 and/or SEQ ID NO:58 and/or (ii) a protein comprising afragment of at least 30 amino acids of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:53 and/or SEQ ID NO:58; (e) anucleic acid molecule having a nucleic acid sequence encoding a CD40protein selected from the group consisting of (i) a protein having anamino acid sequence that is at least about 60 percent identical to anamino acid sequence comprising SEQ ID NO:61 and/or (ii) a proteincomprising a fragment of at least 20 amino acids of an amino acidsequence comprising SEQ ID NO:61; (f) a nucleic acid molecule having anucleic acid sequence encoding a CD154 protein selected from the groupconsisting of (i) a protein having an amino acid sequence that is atleast about 80 percent identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO:65 and/or SEQ ID NO:70, and/or (ii) aprotein comprising a fragment of at least 35 amino acids of an aminoacid sequence selected from the group consisting of SEQ ID NO:65 and/orSEQ ID NO:70; (g) a nucleic acid molecule having a nucleic acid sequenceencoding a CD154 protein selected from the group consisting of (i) aprotein having an amino acid sequence that is at least about 85 percentidentical to an amino acid sequence selected from the group consistingof SEQ ID NO:73 and/or SEQ ID NO:78, and/or (ii) a protein comprising afragment of at least 50 amino acids of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:73 and/or SEQ ID NO:78; (h) anucleic acid molecule having a nucleic acid sequence encoding an IL-5protein selected from the group consisting of (i) a protein having anamino acid sequence that is at least about 85 percent identical to anamino acid sequence selected from the group consisting of SEQ ID NO:81and/or SEQ ID NO:86 and/or (ii) a protein comprising a fragment of atleast 20 amino acids of an amino acid sequence selected from the groupconsisting of SEQ ID NO:81 and/or SEQ ID NO:86; (i) a nucleic acidmolecule having a nucleic acid sequence encoding an IL-13 proteinselected from the group consisting of (i) a protein having an amino acidsequence that is at least about 70 percent identical to an amino acidsequence selected from the group consisting of SEQ ID NO:92, SEQ IDNO:97, SEQ ID NO:100, and/or SEQ ID NO:105 and/or (ii) a proteincomprising a fragment of at least 15 amino acids of an amino acidsequence selected from the group consisting of SEQ ID NO:92, SEQ IDNO:97, SEQ ID NO:100, and/or SEQ ID NO:105; (j) a nucleic acid moleculehaving a nucleic acid sequence encoding an interferon alpha proteinhaving an amino acid sequence that is selected from the group consistingof amino acid sequence SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQID NO:117, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165,SEQ ID NO:168, and/or SEQ ID NO:171; (k) a nucleic acid molecule havinga nucleic acid sequence encoding a GMCSF protein having an amino acidsequence that is selected from the group consisting of amino acidsequence SEQ ID NO:120, SEQ ID NO:125, and/or (1) a nucleic acidmolecule comprising a complement of any of the before-mentioned nucleicacid sequences; wherein said IL-4 protein elicits an immune responseagainst an IL-4 protein selected from the group consisting of SEQ IDNO:2 and/or SEQ ID NO:20 and/or is a protein with interleukin-4activity, said Flt-3 ligand protein elicits an immune response against aFlt-3 ligand protein selected from the group consisting of SEQ ID NO:7,SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:44,and/or SEQ ID NO:49 and/or is a protein with Flt-3 ligand activity, saidCD40 protein elicits an immune response against a CD40 protein selectedfrom the group consisting of SEQ ID NO:53, SEQ ID NO:58, and/or SEQ IDNO:61 and/or is a protein with CD40 activity, said CD154 protein elicitsan immune response against a CD154 protein selected from the groupconsisting of SEQ ID NO:65, SEQ ID NO:70, SEQ ID NO:73, and /or SEQ IDNO:78 and/or is a protein with CD154 activity, said IL-5 protein elicitsan immune response against a IL-5 protein selected from the groupconsisting of SEQ ID NO:81 and/or SEQ ID NO:86 and/or is a protein withIL-5 activity, said IL-13 protein elicits an immune response against anIL-13 protein selected from the group consisting of SEQ ID NO:92, SEQ IDNO:97, SEQ ID NO:100, and/or SEQ ID NO:105 and/or is a protein withIL-13 activity, said interferon alpha protein elicits an immune responseagainst an interferon alpha protein selected from the group consistingof SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ IDNO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168,and/or SEQ ID NO:171 and/or is a protein with interferon alpha activity,and said GMCSF protein elicits an immune response against a GMCSFprotein selected from the group consisting of SEQ ID NO:120 and/or SEQID NO:125 and/or is a protein with GM-CSF activity.

In one embodiment, an IL-4 nucleic acid molecule of the presentinvention encodes a protein that is at least about 85%, preferably atleast about 90%, preferably at least about 92%, and even more preferablyat least about 95% identical to PCaIL-4₁₃₂ and/or PCaIL-4₁₀₈ In oneembodiment, a Flt-3 ligand nucleic acid molecule of the presentinvention encodes a protein that is at least about 75%, even morepreferably at least about 80%, even more preferably at least about 85%,even more preferably at least about 90%, and even more preferably atleast about 95% identical to PCaFlt3L₂₉₄, PCaFlt3L₂₆₈, PCaFlt3L₂₇₆,PCaFlt3L₂₅₀, and/or PCaFlt3L₃₁. In one embodiment, a Flt-3 ligandnucleic acid molecule of the present invention encodes a protein that isat least about 75%, more preferably at least about 80%, even morepreferably at least about 85%, even more preferably at least about 90%,and even more preferably at least about 95% identical to PFeFlt3L₂₉₁,and/or PFeFlt3L₂₆₅. In one embodiment, a CD40 nucleic acid molecule ofthe present invention encodes a protein that is at least aboutPCaCD40₂₇₄, and/or PCaCD40₂₅₅. In one embodiment, a CD40 nucleic acidmolecule of the present invention encodes a protein that is at leastabout 60%, preferably at least about 65%, preferably at least about 70%,preferably at least about 75%, even more preferably at least about 80%,even more preferably at least about 85%, even more preferably at leastabout 90%, and even more preferably at least about 95% identical toPFeCD40₁₁₂. In one embodiment, a CD154 nucleic acid molecule of thepresent invention encodes a protein that is at least about 80%, at leastabout 85%, more preferably at least about 90%, and even more preferablyat least about 95% identical to PCaCD154₂₆₀, and/or PCaCD154₂₁₁. In oneembodiment , a CD154 nucleic acid molecule of the present inventionencodes a protein that is at least about 85%, more preferably at leastabout 90%, and even more preferably at least about 95% identical toPFeCD154₂₆₀, PFeCD154₂₁₁. In one embodiment, an IL-5 nucleic acidmolecule of the present invention encodes a protein that is at leastabout 85%, more preferably at least about 90%, and even more preferablyat least about 95% identical to PCaIL-5₁₃₄, and/or PCaIL-5₁₁₅. In oneembodiment, an IL-13 nucleic acid molecule of the present inventionencodes a protein that is at least about 70%, at least about 75%, atleast about 80%, preferably at least about 85%, more preferably at leastabout 90%, and even more preferably at least about 95% identical toPCaIL-13₁₃₁, PCaIL-13₁₁₁, PCaIL-13₁₃₀, PCaIL-13₁₁₀. Even more preferredis a nucleic acid molecule encoding PCaIL-4₁₃₂, PCaIL-4₁₀₈, PCaFlt3L₂₉₄,PCaFlt3L₂₆₈, PCaFlt3L₂₇₆, PCaFlt3L₂₅₀, PCaFlt3L₃₁, PFeFlt3L₂₉₁,PFeFlt3L265, PCaCD40₂₇₄, PCaCD40₂₅₅, PFeCD40₁₁₂, PCaCD154₂₆₀,PCaCD154₂₁₁, PFeCD154₂₆₀, PFeCD154₂₁₁, PCaIL-5₁₃₄, PCaIL-5₁₁₅,PCaIL-13₁₃₁, PCaIL-13₁₁₁, PCaIL-13₁₃₀, PCaIL-13₁₁₀ and/or an allelicvariant of such a nucleic acid molecule.

In another embodiment, an IL-4 nucleic acid molecule of the presentinvention encodes a protein having an amino acid sequence that is atleast about 85%, preferably at least about 90%, and even more preferablyabout at least about 95% identical to SEQ ID NO:2, SEQ ID NO:20. Thepresent invention also includes an IL-4 nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:2, and/or SEQ ID NO:20,as well as allelic variants of an IL-4 nucleic acid molecule encoding aprotein having these sequences, including nucleic acid molecules thathave been modified to accommodate codon usage properties of the cells inwhich such nucleic acid molecules are to be expressed.

In another embodiment, a Flt-3 ligand nucleic acid molecule of thepresent invention encodes a protein having an amino acid sequence thatis at least about 75%, even more preferably at least about 80%, evenmore preferably at least about 85%, even more preferably at least about90%, and even more preferably at least about 95% identical to SEQ IDNO:7, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:31, and/or SEQ ID NO:34. Thepresent invention also includes a Flt-3 ligand nucleic acid moleculeencoding a protein having at least a portion of SEQ ID NO:7, SEQ IDNO:23, SEQ ID NO:26, SEQ ID NO:31, and/or SEQ ID NO:34, as well asallelic variants of a Flt-3 ligand nucleic acid molecule encoding aprotein having these sequences, including nucleic acid molecules thathave been modified to accommodate codon usage properties of the cells inwhich such nucleic acid molecules are to be expressed.

In another embodiment, a Flt-3 ligand nucleic acid molecule of thepresent invention encodes a protein having an amino acid sequence thatis at least about 75%, more preferably at least about 80%, even morepreferably at least about 85%, even more preferably at least about 90%,and even more preferably at least about 95% identical to SEQ ID NO:44,and/or SEQ ID NO:49. The present invention also includes a Flt-3 ligandnucleic acid molecule encoding a protein having at least a portion ofSEQ ID NO:44, and/or SEQ ID NO:49, as well as allelic variants of aFlt-3 ligand nucleic acid molecule encoding a protein having thesesequences, including nucleic acid molecules that have been modified toaccommodate codon usage properties of the cells in which such nucleicacid molecules are to be expressed.

In another embodiment, a CD40 nucleic acid molecule of the presentinvention encodes a protein having an amino acid sequence that is atleast about 70%, preferably at least about 75%, even more preferably atleast about 80%, even more preferably at least about 85%, even morepreferably at least about 90%, and even more preferably at least about95% identical to SEQ ID NO:53 and/or SEQ ID NO:58. The present inventionalso includes a CD40 nucleic acid molecule encoding a protein having atleast a portion of SEQ ID NO:53 and/or SEQ ID NO:58, as well as allelicvariants of a CD40 nucleic acid molecule encoding a protein having thesesequences, including nucleic acid molecules that have been modified toaccommodate codon usage properties of the cells in which such nucleicacid molecules are to be expressed.

In another embodiment, a CD40 nucleic acid molecule of the presentinvention encodes a protein having an amino acid sequence that is atleast about 60%, preferably at least about 65%, preferably at leastabout 70%, preferably at least about 75%, even more preferably at leastabout 80%, even more preferably at least about 85%, even more preferablyat least about 90%, and even more preferably at least about 95%identical to SEQ ID NO:60. The present invention also includes a CD40nucleic acid molecule encoding a protein having at least a portion ofSEQ ID NO:60, as well as allelic variants of a CD40 nucleic acidmolecule encoding a protein having these sequences, including nucleicacid molecules that have been modified to accommodate codon usageproperties to the cells in which such nucleic acid molecules are to beexpressed.

In another embodiment, a CD154 nucleic acid molecule of the presentinvention encodes a protein having an amino acid sequence that is atleast about at least about 80%, at least about 85%, more preferably atleast about 90%, and even more preferably at least about 95% identicalto SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:67, and/or SEQ ID NO:69. Thepresent invention also includes a CD154 nucleic acid molecule encoding aprotein having at least a portion of SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:67, and/or SEQ ID NO:69, as well as allelic variants of a CD154nucleic acid molecule encoding a protein having these sequences,including nucleic acid molecules that have been modified to accommodatecodon usage properties of the cells in which such nucleic acid moleculesare to be expressed.

In another embodiment, a CD154 nucleic acid molecule of the presentinvention encodes a protein having an amino acid sequence that is atleast about at least about 85%, more preferably at least about 90%, andeven more preferably at least about 95% identical to SEQ ID NO:72, SEQID NO:75, and/or SEQ ID NO:77. The present invention also includes aCD154 nucleic acid molecule encoding a protein having at least a portionof SEQ ID NO:72, SEQ ID NO:75, and/or SEQ ID NO:77, as well as allelicvariants of a CD154 nucleic acid molecule encoding a protein havingthese sequences, including nucleic acid molecules that have beenmodified to accommodate codon usage properties of the cells in whichsuch nucleic acid molecules are to be expressed.

In another embodiment, an IL-5 nucleic acid molecule of the presentinvention encodes a protein having an amino acid sequence that is atleast about at least about 85%, at least about 85%, more preferably atleast about 90%, and even more preferably at least about 95% identicalto SEQ ID NO:80, SEQ ID NO:83, and/or SEQ ID NO:85. The presentinvention also includes an IL-5 nucleic acid molecule encoding a proteinhaving at least a portion of SEQ ID NO:80, SEQ ID NO:83, and/or SEQ IDNO:85, as well as allelic variants of an IL-5 nucleic acid moleculeencoding a protein having these sequences, including nucleic acidmolecules that have been modified to accommodate codon usage propertiesof the cells in which such nucleic acid molecules are to be expressed.

In another embodiment, an IL-13 nucleic acid molecule of the presentinvention encodes a protein having an amino acid sequence that is atleast about at least about 70%, at least about 75%, at least about 80%,preferably at least about 85%, more preferably at least about 90%, andeven more preferably at least about 95% identical to SEQ ID NO:88, SEQID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:99, SEQ ID NO:102, and/or SEQ ID NO:104. The present invention alsoincludes an IL-13 nucleic acid molecule encoding a protein having atleast a portion of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, and/orSEQ ID NO:104, as well as allelic variants of an IL-13 nucleic acidmolecule encoding a protein having these sequences, including nucleicacid molecules that have been modified to accommodate codon usageproperties of the cells in which such nucleic acid molecules are to beexpressed.

In one embodiment, an IL-4 nucleic acid molecule of the presentinvention is at least about 90%, and preferably at least about 95%identical to nCaIL-4₅₄₉. Even more preferred is a nucleic acid moleculecomprising nCaIL-4₅₄₉, nCaIL-4₃₉₆, nCaIL-4₃₂₄, and/or an allelic variantof such a nucleic acid molecule. In another embodiment, a Flt-3 ligandnucleic acid molecule of the present invention is at least about 75%,more preferably at least about 80%, more preferably at least about 85%,more preferably at least about 90% and even more preferably at leastabout 95% identical to nCaFlt3L₁₀₁₃. Even more preferred is a nucleicacid molecule comprising nCaFlt3L₁₀₁₃, nCaFlt3L₈₈₂, nCaFlt3L₈₀₄,nCaFlt3L₈₂₈, nCaFlt3L₉₈₅, nCaFlt3L₁₀₁₉, nCaFlt3L₉₃, and/or nCaFlt3L₇₅₀,and/or an allelic variant of such a nucleic acid molecule. In oneembodiment, a Flt-3 ligand nucleic acid molecule of the presentinvention is at least about 75%, more preferably at least about 80%,more preferably at least about 85%, more preferably at least about 90%and even more preferably at least about 95% identical to nFeFlt3L₉₄₂.Even more preferred is a nucleic acid molecule comprising nFeFlt3L₃₉₅,nFeFlt3L₇₉₃, nFeFlt3L₉₄₂, nFeFlt3L₈₇₃, and/or nFeFlt3L₇₉₅, and/or anallelic variant of such a nucleic acid molecule. In one embodiment, aCD40 nucleic acid molecule of the present invention is at least about70%, at least about 75%, more preferably at least about 80%, morepreferably at least about 85%, more preferably at least about 90% andeven more preferably at least about 95% identical to nCaCD40₃₂₁,nCaCD40₁₄₂₅, nCaCD40₈₂₂, and/or nCaCD40₇₆₅, and/or an allelic variant ofsuch a nucleic acid molecule. In one embodiment, a CD40 nucleic acidmolecule of the present invention is at least about 70%, at least about75%, more preferably at least about 80%, more preferably at least about85%, more preferably at least about 90% and even more preferably atleast about 95% identical to nFeCD40₃₃₆, and/or an allelic variant ofsuch a nucleic acid molecule. In one embodiment, a CD154 nucleic acidmolecule of the present invention is at least about 85%, preferably atleast about 85%, more preferably at least about 90% and even morepreferably at least about 95% identical to nCaCD154₃₉₀, nCaCD154₈₇₈,nCaCD154₇₈₀, and/or nCaCD1541₆₃₃, and/or an allelic variant of such anucleic acid molecule. In one embodiment, a CD154 nucleic acid moleculeof the present invention is at least about 91%, and preferably about 95%identical to nFeCD154₈₈₅, nFeCD154₇₈₀, and/or nFeCD154₆₃₃, and/or anallelic variant of such a nucleic acid molecule. In one embodiment, anIL-5 molecule of the present invention is at least about 90% andpreferably at least about 95% identical to nCaIL-5₆₁₀, nCaIL-5₄₀₂,and/or nCaIL-5₃₄₅, and/or an allelic variant of such a nucleic acidmolecule. In another embodiment, an IL-13 molecule of the presentinvention is at least about 65%, at least about 70%, preferably at leastabout 75%, more preferably at least about 80%, more preferably at leastabout 85%, more preferably at least about 90% and even more preferablyat least about 95% identical to nCaIL-13₁₆₆, nCaIL-13₂₇₂, nCaIL-13₂₇₈,nCaIL-13₁₃₀₂, nCaIL-13₃₉₃, nCaIL-13₃₃₃, nCaIL-13₁₂₆₉, nCaIL-13₃₉₀,and/or nCaIL-13₃₃₀, and/or an allelic variant of such a nucleic acidmolecule.

In another embodiment, an IL-4 nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that is at least about 90%,and preferably at least about 95% identical to SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:19, and/or SEQ ID NO:21. The presentinvention also includes an IL-4 nucleic acid molecule comprising atleast a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:19, and/or SEQ ID NO:21, as well as allelic variants of suchIL-4 nucleic acid molecules, including nucleic acid molecules that havebeen modified to accommodate codon usage properties of the cells inwhich such nucleic acid molecules are to be expressed.

In another embodiment, a Flt-3 ligand nucleic acid molecule of thepresent invention comprises a nucleic acid sequence that is at leastabout 75%, preferably at least about 80%, more preferably at least about85%, more preferably at least about 90% and even more preferably atleast about 95% identical to SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:36, and/or SEQ ID NO:37. The present invention alsoincludes a Flt-3 ligand- nucleic acid molecule comprising at least aportion of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:36, and/or SEQ ID NO:37, as well as allelic variants of such Flt-3ligand nucleic acid molecules, including nucleic acid molecules thathave been modified to accommodate codon usage properties of the cells inwhich such nucleic acid molecules are to be expressed.

In one embodiment, a Flt-3 ligand nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that is at least about 75%,more preferably at least about 80%, more preferably at least about 85%,more preferably at least about 90% and even more preferably at leastabout 95% identical to SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and/or SEQ ID NO:50.The present invention also includes a Flt-3 ligand- nucleic acidmolecule comprising at least a portion of SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,and/or SEQ ID NO:50, as well as allelic variants of such Flt-3 ligandnucleic acid molecules, including nucleic acid molecules that have beenmodified to accommodate codon usage properties of the cells in whichsuch nucleic acid molecules are to be expressed.

In one embodiment, a CD40 nucleic acid molecule of the present inventioncomprises a nucleic acid sequence that is at least about 70%, at leastabout 75%, more preferably at least about 80%, more preferably at leastabout 85%, more preferably at least about 90% and even more preferablyat least about 95% identical to SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and/or SEQ ID NO:59.The present invention also includes a CD40 nucleic acid moleculecomprising at least a portion of SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and/or SEQ ID NO:59, aswell as allelic variants of such CD40 nucleic acid molecules, includingnucleic acid molecules that have been modified to accommodate codonusage properties of the cells in which such nucleic acid molecules areto be expressed.

In one embodiment, a CD40 nucleic acid molecule of the present inventioncomprises a nucleic acid sequence that is at least about 70%, at leastabout 75%, more preferably at least about 80%, more preferably at leastabout 85%, more preferably at least about 90% and even more preferablyat least about 95% identical to SEQ ID NO:60 and/or SEQ ID NO:62. Thepresent invention also includes a CD40 nucleic acid molecule comprisingat least a portion of SEQ ID NO:60 and/or SEQ ID NO:62, as well asallelic variants of such CD40 nucleic acid molecules, including nucleicacid molecules that have been modified to accommodate codon usageproperties of the cells in which such nucleic acid molecules are to beexpressed.

In one embodiment, a CD154 nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that is at least about 85%,preferably at least about 85%, more preferably at least about 90% andeven more preferably at least about 95% identical to SEQ ID NO:63, SEQID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and/orSEQ ID NO:71. The present invention also includes a CD154 nucleic acidmolecule comprising at least a portion of SEQ ID NO:63, SEQ ID NO:64,SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and/or SEQ IDNO:71, as well as allelic variants of such CD154 nucleic acid molecules,including nucleic acid molecules that have been modified to accommodatecodon usage properties of the cells in which such nucleic acid moleculesare to be expressed.

In one embodiment, a CD154 nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that is at least about 91%,and preferably about 95% identical to SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, and/or SEQ ID NO:79. The presentinvention also includes a CD154 nucleic acid molecule comprising atleast a portion of SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, and/or SEQ ID NO:79, as well as allelic variants ofsuch CD154 nucleic acid molecules, including nucleic acid molecules thathave been modified to accommodate codon usage properties of the cells inwhich such nucleic acid molecules are to be expressed.

In one embodiment, an IL-5 nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that is at least about 90%and preferably at least about 95% identical to SEQ ID NO:80, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and/or SEQ ID NO:87.The present invention also includes an IL-5 nucleic acid moleculecomprising at least a portion of SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:85, and/or SEQ ID NO:87, as well asallelic variants of such IL-5 nucleic acid molecules, including nucleicacid molecules that have been modified to accommodate codon usageproperties of the cells in which such nucleic acid molecules are to beexpressed.

In one embodiment, an IL-13 nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that is at least about 65%,at least about 70%, preferably at least about 75%, more preferably atleast about 80%, more preferably at least about 85%, more preferably atleast about 90% and even more preferably at least about 95% identical toSEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93,SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99,SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, and/or SEQID NO:106. The present invention also includes an IL-13 nucleic acidmolecule comprising at least a portion of SEQ ID NO:88, SEQ ID NO:89,SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:94, SEQ ID NO:102,SEQ ID NO:103, SEQ ID NO:104, and/or SEQ ID NO:106, as well as allelicvariants of such IL-13 nucleic acid molecules, including nucleic acidmolecules that have been modified to accommodate codon usage propertiesof the cells in which such nucleic acid molecules are to be expressed.

In one embodiment, an IFNα nucleic acid molecule of the presentinvention is identical to SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:110,SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:116, SEQ IDNO:118, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:160, SEQID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:167,SEQ ID NO:169, SEQ ID NO:170, and/or SEQ ID NO:172.

In another embodiment, a GM-CSF nucleic acid molecule of the presentinvention is identical to SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122,SEQ ID NO:123, SEQ ID NO:124, and/or SEQ ID NO:126.

Knowing the nucleic acid sequences of certain immunoregulatory nucleicacid molecules of the present invention allows one skilled in the artto, for example, (a) make copies of those nucleic acid molecules, (b)obtain nucleic acid molecules including at least a portion of suchnucleic acid molecules (e.g., nucleic acid molecules includingfull-length genes, full-length coding regions, regulatory controlsequences, truncated coding regions), and/or (c) obtain otherimmunoregulatory nucleic acid molecules. Such nucleic acid molecules canbe obtained in a variety of ways including screening appropriateexpression libraries with antibodies of the present invention;traditional cloning techniques using oligonucleotide probes of thepresent invention to screen appropriate libraries; and PCR amplificationof appropriate libraries or DNA using oligonucleotide primers of thepresent invention. Preferred libraries to screen or from which toamplify nucleic acid molecules include mammalian cDNA libraries as wellas genomic DNA libraries. Similarly, preferred DNA sources from which toamplify nucleic acid molecules include mammalian cDNA and genomic DNA.Techniques to clone and amplify genes are disclosed, for example, inSambrook et al., ibid.

The present invention also includes nucleic acid molecules that areoligonucleotides capable of hybridizing, under stringent hybridizationconditions, with complementary regions of other, preferably longer,nucleic acid molecules of the present invention such as those comprisingcanine interleukin-4, canine or feline Flt-3 ligand, canine or felineCD40, canine or feline CD154, canine interleukin-5, canineinterleukin-13, feline interferon alpha, or feline GM-CSF nucleic acidmolecules. Oligonucleotides of the present invention can be RNA, DNA, orderivatives of either. The minimum size of such oligonucleotides is thesize required for formation of a stable hybrid between anoligonucleotide and a complementary sequence on a nucleic acid moleculeof the present invention. A preferred oligonucleotide of the presentinvention has a maximum size of about 100 nucleotides. The presentinvention includes oligonucleotides that can be used as, for example,probes to identify nucleic acid molecules, primers to produce nucleicacid molecules, or therapeutic reagents to inhibit canine interleukin-4,canine or feline Flt-3 ligand, canine or feline CD40, canine or felineCD154, canine interleukin-5, canine interleukin-13, feline interferonalpha, or feline GM-CSF protein production or activity (e.g., asantisense-, triplex formation-, ribozyme- and/or RNA drug-basedreagents). The present invention also includes the use of sucholigonucleotides to protect animals from disease using one or more ofsuch technologies. Appropriate oligonucleotide-containing therapeuticcompositions can be administered to an animal using techniques known tothose skilled in the art.

One embodiment of the present invention includes a recombinant vector,which includes at least one isolated nucleic acid molecule of thepresent invention, inserted into any vector capable of delivering thenucleic acid molecule into a host cell. Such a vector containsheterologous nucleic acid sequences, that is nucleic acid sequences thatare not naturally found adjacent to nucleic acid molecules of thepresent invention and that preferably are derived from a species otherthan the species from which the nucleic acid molecule(s) are derived.The vector can be either RNA or DNA, either prokaryotic or eukaryotic,and typically is a virus or a plasmid. Recombinant vectors can be usedin the cloning, sequencing, and/or otherwise manipulatingimmunoregulatory nucleic acid molecules of the present invention.

One type of recombinant vector, referred to herein as a recombinantmolecule, comprises a nucleic acid molecule of the present inventionoperatively linked to an expression vector. The phrase operativelylinked refers to insertion of a nucleic acid molecule into an expressionvector in a manner such that the molecule is able to be expressed whentransformed into a host cell. As used herein, an expression vector is aDNA or RNA vector that is capable of transforming a host cell and ofeffecting expression of a specified nucleic acid molecule. Preferably,the expression vector is also capable of replicating within the hostcell. Expression vectors can be either prokaryotic or eukaryotic, andare typically viruses or plasmids. Expression vectors of the presentinvention include any vectors that function (i.e., direct geneexpression) in recombinant cells of the present invention, including inbacterial, fungal, parasite, insect, other animal, and plant cells.Preferred expression vectors of the present invention can direct geneexpression in bacterial, yeast, insect and mammalian cells, and morepreferably in the cell types disclosed herein, more preferably in vivo.

In particular, expression vectors of the present invention containregulatory sequences such as transcription control sequences,translation control sequences, origins of replication, and otherregulatory sequences that are compatible with the recombinant cell andthat control the expression of nucleic acid molecules of the presentinvention. In particular, recombinant molecules of the present inventioninclude transcription control sequences. Transcription control sequencesare sequences which control the initiation, elongation, and terminationof transcription. Particularly important transcription control sequencesare those which control transcription initiation, such as promoter,enhancer, operator and repressor sequences. Suitable transcriptioncontrol sequences include any transcription control sequence that canfunction in at least one of the recombinant cells of the presentinvention. A variety of such transcription control sequences are knownto those skilled in the art. Preferred transcription control sequencesinclude those which function in bacterial, yeast, helminth and/or otherendoparasite, insect and mammalian cells, such as, but not limited to,tac, lac, trp, trc, oxy-pro, omp/lpp, rmB, bacteriophage lambda (such aslambda p_(L) and lambda p_(R) and fusions that include such promoters),bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6,bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcoholoxidase, alphavirus subgenomic promoter, antibiotic resistance gene,baculovirus, Haliothis zea insect virus, vaccinia virus, herpesvirus,raccoon poxvirus, other poxvirus, adenovirs, cytomegalovirus (such asimmediate early promoter), simian virus 40, retrovirus, actin,retroviral long terminal repeat, Rous sarcoma virus, heat shock,phosphate and nitrate transcription control sequences as well as othersequences capable of controlling gene expression in prokaryotic oreukaryotic cells. Additional suitable transcription control sequencesinclude tissue-specific promoters and enhancers as well aslymphokine-inducible promoters (e.g., promoters inducible by interferonsor interleukins). Transcription control sequences of the presentinvention can also include naturally occurring transcription controlsequences naturally associated with mammals, such as dog, cat, horse orhuman transcription control sequences.

Suitable and preferred nucleic acid molecules to include in recombinantvectors of the present invention are as disclosed herein. Preferrednucleic acid molecules to include in recombinant vectors, andparticularly in recombinant molecules, include nCaIL-4₅₄₉, nCaIL-4₃₉₆,nCaIL-4₃₂₄, nCaFlt3L₁₀₁₃, nCaFlt3L₈₈₂, nCaFlt3L₈₀₄, nCaFlt3L₈₂₈,nCaFlt3L₉₈₅, nCaFlt3L₁₀₁₉, nCaFlt3L₉₃, nCaFlt3L₇₅₀, nFeFlt3L₃₉₅,nFeFlt3L₇₉₃, nFeFlt3L₉₄₂, nFeFlt3L₈₇₃, nFeFlt3L₇₉₅, nCaCD40₃₂₁,nCaCD40₁₄₂₅, nCaCD40₈₂₂, nCaCD40₇₆₅, nFeCD40₃₃₆, nCaCD154₃₉₀,nCaCD1541₈₇₈, nCaCD154₇₈₀, nCaCD154₆₃₃, nFeCD154₈₈₅, nFeCD154₇₈₀,nFeCD154₆₃₃, nCaIL-5₆₁₀, nCaIL-5₄₀₂, nCaIL-5₃₄₅, nCaIL-13₁₆₆,nCaIL-13₂₇₂, nCaIL-13₂₇₈, nCaIL-13₁₃₀₂, nCaIL-13₃₉₃, nCaIL-13₃₃₃,nCaIL-13₁₂₆₉, nCaIL-13₃₉₀, nCaIL-13₃₃₀, nFeIFNα_(567a), nFeIFNα_(567b),nFeIFNα_(567c), nFeIFNα_(498a), nFeIFNα_(498b), nFeIFNα_(498c),nFeIFNα_(582d), nFeIFNα_(513d), nFeIFNα_(567e), nFeIFNα_(498e),nFeGMCSF₄₄₄, nFeGMCSF₄₃₂, and/or nFeGMCSF₃₈₁.

Recombinant molecules of the present invention may also (a) containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed parasitic helminth protein of the present inventionto be secreted from the cell that produces the protein and/or (b)contain fuision sequences which lead to the expression of nucleic acidmolecules of the present invention as fusion proteins. Examples ofsuitable signal segments include any signal segment capable of directingthe secretion of a protein of the present invention. Preferred signalsegments include, but are not limited to, tissue plasminogen activator(t-PA), interferon, interleukin, growth hormone, histocompatibility andviral envelope glycoprotein signal segments. Suitable fusion segmentsencoded by fusion segment nucleic acids are disclosed herein. Inaddition, a nucleic acid molecule of the present invention can be joinedto a fusion segment that directs the encoded protein to the proteosome,such as a ubiquitin fusion segment. Eukaryotic recombinant molecules mayalso include intervening and/or untranslated sequences surroundingand/or within the nucleic acid sequences of nucleic acid molecules ofthe present invention.

Another embodiment of the present invention includes a recombinant cellcomprising a host cell transformed with one or more recombinantmolecules of the present invention. Transformation of a nucleic acidmolecule into a cell can be accomplished by any method by which anucleic acid molecule can be inserted into the cell. Transformationtechniques include, but are not limited to, transfection,electroporation, microinjection, lipofection, adsorption, and protoplastfusion. A recombinant cell may remain unicellular or may grow into atissue, organ or a multicellular organism. Transformed nucleic acidmolecules of the present invention can remain extrachromosomal or canintegrate into one or more sites within a chromosome of the transformed(i.e., recombinant) cell in such a manner that their ability to beexpressed is retained. Preferred nucleic acid molecules with which totransform a cell include immunoregulatory nucleic acid molecules of thepresent invention disclosed herein. Particularly preferred nucleic acidmolecules with which to transform to cell include nCaIL-4₅₄₉,nCaIL-4₃₉₆, nCaIL-4₃₂₄, nCaFlt3L₁₀₁₃, nCaFtl3L₈₈₂, nCaFlt3L₈₀₄,nCaFtl3₈₂₈, nCaFlt3L₉₈₅, nCaFtl3L₁₀₁₉, nCaFlt3L₉₃, nCaFlt3L₇₅₀,nFeFlt3L₃₉₅, nFeFlt3L₇₉₃, nFeFlt3L₉₄₂, nFeFlt3L₈₇₃, nFeFlt3L₇₉₅,nCaCD40₃₂₁, nCaCD40₁₄₂₅, nCaCD40₈₂₂, nCaCD40₇₆₅, nFeCD40₃₃₆,nCaCD154₃₉₀, nCaCD1541₈₇₈, nCaCD154₇₈₀, nCaCD154₆₃₃, nFeCD154₈₈₅,nFeCD154₇₈₀, nFeCD154₆₃₃, nCaIL-5₆₁₀, nCaIL-5₄₀₂, nCaIL-5₃₄₅,nCaIL-13₁₆₆, nCaIL-13₂₇₂, nCaIL-13₂₇₈, nCaIL-13₁₃₀₂, nCaIL-13₃₉₃,nCaIL-13₃₃₃, nCaIL-13₁₂₆₉, nCaIL-13₃₉₀, nCaIL-13₃₃₀, nFeIFNα_(567a),nFeIFNα_(567b), nFeIFNα_(567c), nFeIFNα_(498a), nFeIFNα_(498b),nFeIFNα_(498c), nFeIFNα_(528d), nFeIFNα_(513d), nFeIFNα_(567c),nFeIFNα_(498e), nFeGMCSF₄₄₄, nFeGMCSF₄₃₂, and/or nFeGMCSF₃₈₁.

Suitable host cells to transform include any cell that can betransformed with a nucleic acid molecule of the present invention. Hostcells can be either untransformed cells or cells that are alreadytransformed with at least one nucleic acid molecule (e.g., nucleic acidmolecules encoding one or more proteins of the present invention and/orother proteins useful in the production of multivalent vaccines). Hostcells of the present invention either can be endogenously (i.e.,naturally) capable of producing immunoregulatory proteins of the presentinvention or can be capable of producing such proteins after beingtransformed with at least one nucleic acid molecule of the presentinvention. Host cells of the present invention can be any cell capableof producing at least one protein of the present invention, and includebacterial, fingal (including yeast), parasite (including helminth,protozoa and endoparasite), other insect, other animal and plant cells.Preferred host cells include bacterial, mycobacterial, yeast, helminth,insect and mammalian cells. More preferred host cells includeSalmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera,Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells(Madin-Darby canine kidney cell line), CRFK cells (Crandell felinekidney cell line), CV-1 cells (African monkey kidney cell line used, forexample, to culture raccoon poxvirus), COS (e.g., COS-7) cells, chinesehamster ovary (CHO) cells, Ltk cells and Vero cells. Particularlypreferred host cells are Escherichia coli, including E. coli K-12derivatives; Salmonella typhi; Salmonella typhimurium, includingattenuated strains such as UK-1 ₀3987 and SR-11 ₀4072; Spodopterafrugiperda; Trichoplasia ni; BHK cells; MDCK cells; CRFK cells; CV-1cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hostsinclude other kidney cell lines, other fibroblast cell lines (e.g.,human, murine or chicken embryo fibroblast cell lines), myeloma celllines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK³¹ cellsand/or HeLa cells. In one embodiment, the proteins may be expressed asheterologous proteins in myeloma cell lines employing immunoglobulinpromoters.

A recombinant cell is preferably produced by transforming a host cellwith one or more recombinant molecules, each comprising one or morenucleic acid molecules of the present invention operatively linked to anexpression vector containing one or more transcription controlsequences, examples of which are disclosed herein.

A recombinant cell of the present invention includes any celltransformed with at least one of any nucleic acid molecule of thepresent invention. Suitable and preferred nucleic acid molecules as wellas suitable and preferred recombinant molecules with which to transfercells are disclosed herein.

Recombinant cells of the present invention can also be co-transformedwith one or more recombinant molecules including any of canineinterleukin-4, canine or feline Flt3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF nucleic acid molecule encodingone or more proteins of the present invention and/or one or more othernucleic acid molecules encoding other therapeutic compounds, asdisclosed herein (e.g., to produce multivalent vaccines).

Recombinant DNA technologies can be used to improve expression oftransformed nucleic acid molecules by manipulating, for example, thenumber of copies of the nucleic acid molecules within a host cell, theefficiency with which those nucleic acid molecules are transcribed, theefficiency with which the resultant transcripts are translated, and theefficiency of post-translational modifications. Recombinant techniquesuseful for increasing the expression of nucleic acid molecules of thepresent invention include, but are not limited to, operatively linkingnucleic acid molecules to high-copy number plasmids, integration of thenucleic acid molecules into one or more host cell chromosomes, additionof vector stability sequences to plasmids, substitutions ormodifications of transcription control signals (e.g., promoters,operators, enhancers), substitutions or modifications of translationalcontrol signals (e.g., ribosome binding sites, Shine-Dalgamo sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, deletion of sequencesthat destabilize transcripts, and use of control signals that temporallyseparate recombinant cell growth from recombinant enzyme productionduring fermentation. The activity of an expressed recombinant protein ofthe present invention may be improved by fragmenting, modifying, orderivatizing nucleic acid molecules encoding such a protein.

Isolated immunoregulatory proteins of the present invention can beproduced in a variety of ways, including production and/or recovery ofnatural proteins, production and/or recovery of recombinant proteins,and/or chemical synthesis of the proteins. In one embodiment, anisolated protein of the present invention is produced by culturing acell capable of expressing the protein under conditions effective toproduce the protein, and recovering the protein. A preferred cell toculture is a recombinant cell of the present invention. Effectiveculture conditions include, but are not limited to, effective media,bioreactor, temperature, pH and oxygen conditions that permit proteinproduction. An effective medium refers to any medium in which a cell iscultured to produce an immunoregulatory protein of the presentinvention. Such medium typically comprises an aqueous medium havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes, and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantproteins of the present invention may either remain within therecombinant cell; be secreted into the fermentation medium; be secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or be retained on the outer surface of a cell or viralmembrane.

The phrase “recovering the protein”, as well as similar phrases, refersto collecting the whole fermentation medium containing the protein andneed not imply additional steps of separation or purification. Proteinsof the present invention can be purified using a variety of standardprotein purification techniques, such as, but not limited to, affinitychromatography, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, gel filtrationchromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and/or differential solubilizaion.Proteins of the present invention are preferably retrieved in“substantially pure” form. As used herein, “substantially pure” refersto a purity that allows for the effective use of the protein as atherapeutic composition or diagnostic. A therapeutic composition foranimals, for example, should exhibit no substantial toxicity andpreferably should be capable of stimulating the production of antibodiesin a treated animal.

The present invention also includes isolated (i.e., removed from theirnatural milieu) antibodies that selectively bind to an immunoregulatoryprotein of the present invention and/ora mimetope thereof (e.g.,anti-IL-4 antibodies, anti-Flt-3 ligand antibodies, anti-CD40antibodies, anti-CD154 antibodies, anti-IL-5 antibodies, anti-IL-13antibodies, anti-IFNα antibodies, and/or anti-GM-CSF antibodies). Asused herein, the term “selectively binds to” an immunoregulatory proteinof the present invention, refers to the ability of antibodies of thepresent invention to preferentially bind to specified proteins and/ormimetopes thereof of the present invention. Binding can be measuredusing a variety of methods standard in the art including enzymeimmunoassays (e.g., ELISA), immunoblot assays, etc.; see, for example,Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, aLaboratory Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid.,is incorporated by this reference herein in its entirety. An anti-IL-4antibody of the present invention preferably selectively binds to anIL-4 protein in such a way as to inhibit the function of that protein.An anti-Flt-3 ligand antibody of the present invention preferablyselectively binds to a Flt-3 ligand- protein in such a way as to inhibitthe function of that protein. An anti-CD40 antibody of the presentinvention preferably selectively binds to a CD40 protein in such a wayas to inhibit the function of that protein. An anti-CD154 antibody ofthe present invention preferably selectively binds to a CD154 protein insuch a way as to inhibit the function of that protein. An anti-IL-5antibody of the present invention preferably selectively binds to anIL-5 protein in such a way as to inhibit the function of that protein.An anti-IL-13 antibody of the present invention preferably selectivelybinds to an IL-13 protein in such a way as to inhibit the function ofthat protein. An anti-IFNα antibody of the present invention preferablyselectivity binds to an IFNα protein in such a way as to inhibit thefunction of that protein. An anti-GM-CSF antibody of the presentinvention preferably selectively binds to a GM-CSF protein in such a wayas to inhibit the function of that protein.

Isolated antibodies of the present invention can include antibodies inserum, or antibodies that have been purified to varying degrees.Antibodies of the present invention can be polyclonal or monoclonal, orcan be functional equivalents such as antibody fragments and/orgenetically-engineered antibodies, including single chain antibodies orchimeric antibodies that can bind to one or more epitopes.

A preferred method to produce antibodies of the present inventionincludes (a) administering to an animal an effective amount of aprotein, peptide and/ormimetope thereof of the present invention toproduce the antibodies and (b) recovering the antibodies. In anothermethod, antibodies of the present invention are produced recombinantlyusing techniques as heretofore disclosed to produce any of theimmunoregulatory proteins of the present invention. Antibodies raisedagainst defined proteins or mimetopes can be advantageous because suchantibodies are not substantially contaminated with antibodies againstother substances that might otherwise cause interference in a diagnosticassay or side effects if used in a therapeutic composition.

Antibodies of the present invention have a variety of potential usesthat are within the scope of the present invention. For example, suchantibodies can be used (a) as reagents in assays to detect animmunoregulatory protein of the present invention, (b) as reagents inassays to modulate cellular activity through an immunoregulatory proteinof the present invention (e.g., mimicking ligand binding to a canineinterleukin-4, canine or feline Flt-3 ligand, canine or feline CD40,canine or feline CD154, canine interleukin-5, canine interleukin-13,feline interferon alpha, or feline GM-CSF protein, as appropriate),and/or (c) as tools to screen expression libraries and/or to recoverdesired proteins of the present invention from a mixture of proteins andother contaminants. Furthermore, antibodies of the present invention canbe used to target compounds (e.g., nucleic acid molecules, drugs orproteins) to antigen presenting cells. Targeting can be accomplished byconjugating (i.e., stably joining) such antibodies to the compoundsusing techniques known to those skilled in the art. Suitable compoundsare known to those skilled in the art.

One embodiment of the present invention is a therapeutic compositionthat, when administered to an animal in an effective manner, is capableof regulating an immune response in an animal. Therapeutic compositionsof the present invention can include at least one of the followingtherapeutic compounds: an isolated IL-4, Flt-3 ligand, CD40, CD154,IL-5, IL-13, IFNα, and/or GM-CSF protein of the present invention and/ora mimetope thereof; an isolated IL-4, Flt-3 ligand, CD40, CD154, IL-5,IL-13, IFNα, and/or GM-CSF nucleic acid molecule of the presentinvention; an isolated antibody that selectively binds to an IL-4, Flt-3ligand, CD40, CD154, IL-5, IL-13, IFNα, and/or GM-CSF protein of thepresent invention; an inhibitor of canine IL-4, Flt-3 ligand, CD40,CD154, IL-5, IL-1 3, IFNα, and/or GM-CSF function identified by itsability to bind to an IL-4, Flt-3 ligand, CD40, CD154, IL-5, IL-13,IFNα, and/or GM-CSF protein, respectively, of the present invention;such an inhibitor can inhibit binding of the respective immunoregulatoryprotein with its respective receptor, or inhibit the activity therespective protein. Methods to perform such assays to measure bindingand/or activity of an immunoregulatory protein of the present inventionare known to those of skill in the art, and are described, for example,in Janeway, et al., ibid. As used herein, a therapeutic compound refersto a compound that, when administered to an animal in an effectivemanner, is able to treat, ameliorate, and/or prevent a disease. Examplesof proteins, nucleic acid molecules, antibodies and/or inhibitors of thepresent invention are disclosed herein.

The present invention also includes a therapeutic composition comprisingat least one IL-4-, Flt-3 ligand-, CD40-, CD154-, IL-5-, IL-13-, IFNα-,and/or GM-CSF-based compound of the present invention in combinationwith at least one additional therapeutic compound. Examples of suchcompounds are disclosed herein.

Therapeutic compositions of the present invention can be administered toany animal susceptible to such therapy, preferably to mammals, and morepreferably to dogs, cats, humans, ferrets, horses, cattle, sheep and/orother pets, economic food animals and/or zoo animals. Preferred animalsinclude dogs, cats, horses and/or humans.

A therapeutic composition of the present invention is administered to ananimal in an effective manner such that the composition is capable ofregulating an immune response in that animal. Therapeutic compositionsof the present invention can be administered to animals prior to onsetof a disease (i.e., as a preventative vaccine) and/or can beadministered to animals after onset of a disease in order to treat thedisease (i.e., as a therapeutic vaccine). Preferred diseases to preventand/or treat include autoimmune diseases, allergic reactions, infectiousdiseases, tumor development, inflammatory diseases and/or graftrejection. In one embodiment, a therapeutic composition of the presentinvention is administered with an antigen to enhance an immune responseagainst that antigen.

Therapeutic compositions of the present invention can be formulated inan excipient that the animal to be treated can tolerate. Examples ofsuch excipients include water, saline, Ringer's solution, dextrosesolution, Hank's solution, and/or other aqueous physiologically balancedsalt solutions. Non-aqueous vehicles, such as fixed oils, sesame oil,ethyl olate, or triglycerides may also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and/or Tris buffer, while examplesof preservatives include thimerosal, o-cresol, formalin and/or benyzlalcohol. Standard formulations can either be liquid injectables orsolids which can be taken up in a suitable liquid as a suspension orsolution for injection. Thus, in a non-liquid formulation, the excipientcan comprise dextrose, human serum albumin, preservative, etc., to whichsterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic compositioncan include an adjuvant. Adjuvants are agents that are capable ofenhancing the immune response of an animal to a specific antigen.Suitable adjuvants include, but are not limited to, cytokines,chemokines, and/or compounds that induce the production of cytokinesand/or chemokines (e.g., granulocyte macrophage colony stimulatingfactor (GM-CSF), granulocyte colony stimulating factor (G-CSF),macrophage colony stimulating factor (M-CSF), colony stimulating factor(CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3),interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7),interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12),interferon gamma, interferon gamma inducing factor I (IGIF),transforming growth factor beta, RANTES (regulated upon activation,normal T cell expressed and presumably secreted), macrophageinflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmaniaelongation initiating factor (LEIF)); bacterial components (e.g.,endotoxins, in particular superantigens, exotoxins and cell wallcomponents); aluminum-based salts; calcium-based salts; silica;polynucleotides, toxoids; serum proteins, viral coat proteins; blockcopolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc.Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc.,Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A(Superfos Biosector A/S, Denmark). Protein adjuvants of the presentinvention can be delivered in the form of the protein themselves or ofnucleic acid molecules encoding such proteins using the methodsdescribed herein.

In one embodiment of the present invention, a therapeutic compositioncan include a carrier. Carriers include compounds that increase thehalf-life of a therapeutic composition in the treated animal. Suitablecarriers include, but are not limited to, polymeric controlled releasevehicles, biodegradable implants, liposomes, bacteria, viruses, othercells, oils, esters, and gylcols.

One embodiment of the present invention is a controlled releaseformulation that is capable of slowly releasing a composition of thepresent invention into an animal. As used herein, a controlled releaseformulation comprises a composition of the present invention in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon administration to ananimal, form a solid or a gel in situ. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention iscapable of releasing a composition of the present invention into theblood of the treated animal at a constant rate sufficient to attaintherapeutic dose levels of the composition to regulate an immuneresponse in an animal. The therapeutic composition is preferablyreleased over a period of time ranging from about 1 to about 12 months.A controlled release formulation of the present invention is capable ofeffecting a treatment preferably for at least about 1 month, morepreferably for at least about 3 months, even more preferably for atleast about 6 months, even more preferably for at least about 9 months,and even more preferably for at least about 12 months.

Therapeutic compositions of the present invention can be administered toanimals prior to and/or after onset of disease. Acceptable protocols toadminister therapeutic compositions in an effective manner includeindividual dose size, number of doses, frequency of dose administration,and/or mode of administration. Determination of such protocols can beaccomplished by those skilled in the art. A suitable single dose is adose that is capable of regulating the immune response in an animal whenadministered one or more times over a suitable time period. For example,a preferred single dose of a protein, mimetope or antibody therapeuticcomposition is from about 1 microgram (μg) to about milligrams (mg) ofthe therapeutic composition per kilogram body weight of the animal.Booster vaccinations can be administered from about 2 weeks to severalyears after the original administration. Booster administrationspreferably are administered when the immune response of the animalbecomes insufficient to protect the animal from disease. A preferredadministration schedule is one in which from about 10 μg to about 1 mgof the therapeutic composition per kg body weight of the animal isadministered from about one to about two times over a time period offrom about 2 weeks to about 12 months. Modes of administration caninclude, but are not limited to, subcutaneous, intradermal, intravenous,intranasal, intraoccular, oral, transdermal and/or intramuscular routes.

According to one embodiment, a nucleic acid molecule of the presentinvention can be administered to an animal in a fashion to enableexpression of that nucleic acid molecule into a therapeutic protein ortherapeutic RNA (e.g., antisense RNA, ribozyme, triple helix forms orRNA drug) in the animal. Nucleic acid molecules can be delivered to ananimal in a variety of methods including, but not limited to, (a)administering a naked (i.e., not packaged in a viral coat or cellularmembrane) nucleic acid as a genetic vaccine (e.g., as naked DNA or RNAmolecules, such as is taught, for example in Wolff et al., 1990, Science247, 1465-1468) or (b) administering a nucleic acid molecule packaged asa recombinant virus vaccine or as a recombinant cell vaccine (i.e., thenucleic acid molecule is delivered by a viral or cellular vehicle).

A genetic (i.e., naked nucleic acid) vaccine of the present inventionincludes a nucleic acid molecule of the present invention and preferablyincludes a recombinant molecule of the present invention that preferablyis replication, or otherwise amplification, competent. A genetic vaccineof the present invention can comprise one or more nucleic acid moleculesof the present invention in the form of, for example, a dicistronicrecombinant molecule. Preferred genetic vaccines include at least aortion of a viral genome (i.e., a viral vector). Preferred viral vectorsinclude those based on alphaviruses, poxviruses, adenoviruses,herpesviruses, picomaviruses, and/or retroviruses, with those based onalphaviruses (such as sindbis or Semlikiforest virus), species-specificherpesviruses and/or poxviruses being particularly preferred. Anysuitable transcription control sequence can be used, including thosedisclosed as suitable for protein production. Particularly preferredtranscription control sequences include cytomegalovirus immediate early(preferably in conjunction with Intron-A), Rous sarcoma virus longterminal repeat, and tissue-specific transcription control sequences, aswell as transcription control sequences endogenous to viral vectors ifviral vectors are used. The incorporation of a “strong” polyadenylationsignal is also preferred.

Genetic vaccines of the present invention can be administered in avariety of ways, with intramuscular, subcutaneous, intradermal,transdermal, intranasal and/or oral routes of administration beingpreferred. A preferred single dose of a genetic vaccine ranges fromabout 1 nanogram (ng) to about 600 μg, depending on the route ofadministration and/or method of delivery, as can be determined by thoseskilled in the art. Suitable delivery methods include, for example, byinjection, as drops, aerosolized and/or topically. Genetic vaccines ofthe present invention can be contained in an aqueous excipient (e.g.,phosphate buffered saline) alone or in a carrier (e.g., lipid-basedvehicles).

A recombinant virus vaccine of the present invention includes arecombinant molecule of the present invention that is packaged in aviral coat and that can be expressed in an animal after administration.Preferably, the recombinant molecule is packaging- orreplication-deficient and/or encodes an attenuated virus. A number ofrecombinant viruses can be used, including, but not limited to, thosebased on alphaviruses, poxviruses, adenoviruses, herpesviruses,picomaviruses, and/or retroviruses. Preferred recombinant virus vaccinesare those based on alphaviruses (such as Sindbis virus), raccoonpoxviruses, species-specific herpesviruses and/or species-specificpoxviruses. An example of methods to produce and use alphavirusrecombinant virus vaccines are disclosed in U.S. Pat. No. 5,766, 602 byXiong et al., issued Jun. 16, 1998, which is incorporated by thisreference herein in its entirety.

When administered to an animal, a recombinant virus vaccine of thepresent invention infects cells within the immunized animal and directsthe production of a therapeutic protein or RNA nucleic acid moleculethat is capable of protecting the animal from disease cased by aparasitic helminth as disclosed herein. For example, a recombinant virusvaccine comprising an immunoregulatory nucleic acid molecule of thepresent invention is administered according to a protocol that resultsin the regulation of an immune response in an animal. A preferred singledose of a recombinant virus vaccine of the present invention is fromabout 1×10⁴ to about 1×10⁸ virus plaque forming units (pfu) per kilogrambody weight of the animal. Administration protocols are similar to thosedescribed herein for protein-based vaccines, with subcutaneous,intramuscular, intranasal, intraoccular and/or oral administrationroutes being preferred.

A recombinant cell vaccine of the present invention includes recombinantcells of the present invention that express at least one protein of thepresent invention. Preferred recombinant cells for this embodimentinclude Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda,yeast, (including Saccharomyces cerevisiae and Pichia pastoris), BHK,CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK recombinantcells. Recombinant cell vaccines of the present invention can beadministered in a variety of ways but have the advantage that they canbe administered orally, preferably at doses ranging from about 10⁸ toabout 10¹² cells per kilogram body weight. Administration protocols aresimilar to those described herein for protein-based vaccines.Recombinant cell vaccines can comprise whole cells, cells stripped ofcell walls or cell lysates.

The efficacy of a therapeutic composition of the present invention toregulate the immune response in an animal can be tested in a variety ofways including, but not limited to, detection of cellular immunitywithin the treated animal, determining lymphocyte or dendritic cellactivity, detection of immunoglobulin levels, determining hematopoieticstem cell or hematopoietic early progenitor cell development,determining dendritic cell development or challenge of the treatedanimal with an infectious agent to determine whether the treated animalis resistant to disease. In one embodiment, therapeutic compositions canbe tested in animal models such as mice. Such techniques are known tothose skilled in the art.

One embodiment of the present invention is an inhibitory compound.Preferably, such an inhibitory compound is derived from an IL-4, Flt-3ligand, CD40, CD154, IL-5, IL-13, IFNα, and/or GM-CSF protein of thepresent invention. Examples of inhibitory compounds include an antibodyof the present invention, that is administered to an animal in aneffective manner (i.e., is administrated in an amount so as to bepresent in the animal at a titer that is sufficient, upon interaction ofthat antibody with a native IL-4, Flt-3 ligand, CD40, CD154, IL-5,IL-13, IFNα, and/or GM-CSF protein, to decrease the activity of suchproteins in an animal, at least temporarily). Oligonucleotide nucleicacid molecules of the present invention can also be administered in aneffective manner, thereby reducing expression of either an IL-4, Flt-3ligand, CD40, CD154, IL-5, IL-13, IFNα, and/or GM-CSF protein, in orderto interfere with the protein activity targeted in accordance with thepresent invention. Peptides of an IL-4, Flt-3 ligand, CD40, CD154, IL-5,IL-13, IFNα, and/or GM-CSF protein of the present invention can also beadministered in an effective manner, thereby reducing binding of IL-4,Flt-3 ligand, CD40, CD154, IL-5, IL-13, IFNα, and/or GM-CSF proteins tothe appropriate receptor, in order to interfere with the proteinactivity targeted in accordance with the present invention. Aninhibitory compound of an IL-4, Flt-3 ligand, CD40 , CD154, IL-5, IL-13,IFNα, and/or GM-CSF function can be identified using IL-4, Flt-3 ligand,CD40, CD154, IL-5, IL-13, IFNα, and/or GM-CSF proteins of the presentinvention, respectively.

One embodiment of the present invention is a method to identify acompound capable of inhibiting IL-4 function. Such a method includes thesteps of: (a) contacting (e.g., combining, mixing) an isolated IL-4protein of the present invention, with a putative inhibitory compoundunder conditions in which, in the absence of the compound, the IL-4protein binds to IL-4 receptor or stimulates T cells in a T cellproliferation assay, and (b) determining if the putative inhibitorycompound inhibits the binding of IL-4 protein to IL-4 receptor or thestimulation of T cells in a T cell proliferation assay. Anotherembodiment of the present invention is a method to identify a compoundcapable of inhibiting Flt-3 ligand function. Such a method includes thesteps of: (a) contacting an isolated Flt-3 ligand protein of the presentinvention, with a putative inhibitory compound under conditions inwhich, in the absence of the compound, the Flt-3 ligand protein binds toFlt-3 receptor or stimulates dendritic precursor cells in aproliferation assay, and (b) determining if the putative inhibitorycompound inhibits the binding of Flt-3 ligand protein to Flt-3 receptoror the stimulation of dendritic precursor cells in a proliferationassay. Another embodiment of the present invention is a method toidentify a compound capable of inhibiting CD40 function. Such a methodincludes the steps of (a) contacting an isolated CD40 protein of thepresent invention, with a putative inhibitory compound under conditionsin which, in the absence of the compound, the CD40 protein binds to aCD40 binding partner (e.g., CD154) and (b) determining if the putativeinhibitory compound inhibits the binding of CD40 protein to the CD40binding partner. A CD40 binding partner is a molecule that selectivelybinds to CD40 protein. Likewise, a binding partner for any otherimmunoregulatory protein of the present invention includes moleculesthat selectively bind to that particular immunoregulatory protein.Another embodiment of the present invention is a method to identify acompound capable of inhibiting CD154 function. Such a method includesthe steps of (a) contacting an isolated CD154 protein of the presentinvention, with a putative inhibitory compound under conditions inwhich, in the absence of the compound, the CD154 protein binds to aCD154 binding partner (e.g., CD40) and (b) determining if the putativeinhibitory compound inhibits the binding of CD154 protein to the CD154binding partner. Yet another embodiment of the present invention is amethod to identify a compound capable of inhibiting IL-5 function. Sucha method includes the steps of: (a) contacting an isolated IL-5 proteinof the present invention, with a putative inhibitory compound underconditions in which, in the absence of the compound, the IL-5 proteinbinds to IL-5 receptor or stimulates T cells in a T cell proliferationassay, and (b) determining if the putative inhibitory compound inhibitsthe binding of IL-5 protein to IL-5 receptor or the stimulation of Tcells in a T cell proliferation assay. Another embodiment of the presentinvention is a method to identify a compound capable of inhibiting IL-13function. Such a method includes the steps of: (a) contacting anisolated IL-13 protein of the present invention, with a putativeinhibitory compound under conditions in which, in the absence of thecompound, the IL-13 protein binds to IL-13 receptor or stimulates Tcells in a T cell proliferation assay, and (b) determining if theputative inhibitory compound inhibits the binding of IL-13 protein toIL-13 receptor or the stimulation of T cells in a T cell proliferationassay. Another embodiment of the present invention is a method toidentify a compound capable of inhabiting IFNα function. Such a methodincludes the steps of: (a) contacting an isolated IFNα protein of thepresent invention, with a putative inhibitory compound under conditionsin which, in the absence of the compound; the IFNA protein binds to IFNαreceptor or inhibits proliferation of GM-CSF stimulated TF-1 cells, and(b) determining if the putative inhibitory compound inhibits the bindingof IFNα protein to IFNα receptor or inhibits proliferation of GM-CSFstimulated TF-1 cells. Another embodiment of the present invention is amethod to identify a compound capable of inhibiting GM-CSF function.Such a method includes the steps of: (a) contacting an isolated GM-CSFprotein of the present invention, with a putative inhibitory compoundunder conditions in which, in the absence of said compound, the GM-CSFprotein binds to GM-CSF receptor or stimulates T cells in a T cellproliferation assay, and (b) determining if the putative inhibitorycompound inhibits the binding of GM-CSF protein to GM-CSF receptor orthe stimulation of T cells in a T cell proliferation assay.

Putative inhibitory compounds to screen include small organic molecules,antibodies (including mimetopes thereof), and/or ligand analogs. Suchcompounds are also screened to identify those that are substantially nottoxic in host animals.

Preferred IL-4, Flt-3 ligand, CD40, CD154, IL-5, IL-1 3, IFNα, and/orGM/CSF, proteins in inhibit are those produced by dogs, cats, horses orhumans, even more preferred IL-4, Flt-3 ligand, CD40, CD154, IL-5,IL-13, IFNα, and/or GM-CSF proteins to inhibit are those produced bydomestic dogs or cats. A particularly preferred inhibitor of the presentinvention is capable of regulating an immune response in an animal. Itis also within the scope of the present invention to use inhibitors ofthe present invention to target diseases involving undesired immuneactivity in animals. Compositions comprising inhibitors of IL4, Flt-3ligand, CD40, CD154, IL-5, IL13, IFNα, and/or GM-CSF function can beadministered to animals in an effective manner to regulate the immuneresponse in the animals, and preferably to prevent autoimmune disease,allergy, infectious disease, inflammation or prevent graft rejection inanimals, or to treat animals with such diseases. Effective amountsand/or dosing regimens can be determined using techniques known to thoseskilled in the art.

It is also within the scope of the present invention to use isolatedproteins, mimetopes, nucleic acid molecules and/or antibodies of thepresent invention as diagnostic reagents. Methods to use such diagnosticreagents are well known to those skilled in the art, see, for example,Janeway, et al., ibid., and/or PCT Publication No. WO 98/23964,published Jun. 4, 1998.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES

It is to be noted that the examples include a number of molecularbiology, microbiology, immunology and biochemistry techniques consideredto be familiar to those skilled in the art. Disclosure of suchtechniques can be found, for example, in Sambrook et al., ibid. andAusubel, et al., 1993, Current Protocols in Molecular Biology,Greene/Wiley Interscience, New York, N.Y., and related references.Ausubel, et al, ibid, is incorporated by reference herein in itsentirety.

Example 1

This example describes the insolation and sequencing of canineinterleukin-4 (IL-4) nucleic acid molecules of the present invention.This example also describes expression of recombinant canine IL-4 in E.coli and mammalian cells; development of monoclonal and polyclonalantibodies to E. coli expressed canine IL-4; and bioactivity ofmammalian-expressed and E. coli-expressed canine IL-4.

A. Isolation and Sequencing of a Canine IL-4 Nucleic Acid Molecule.

Initial attempts to isolate a canine IL-4 nucleic acid molecule usingvarious primers corresponding to putative conserved regions of IL-4nucleic acid molecules failed. Forward and reverse primers were thendesigned using a sequence tag site (IL-4sts) described by Venta et al.in GenBank. The forward primer was designated as EL-4stsA, having thenucleic acid sequence 5′ CTATTAATGG GTCTCACCTC CCAA CT 3′, designatedherein as SEQ ID NO:11. The reverse primer was designated as prIL-4stsB,having the nucleic acid sequence 5′ TCAACTCGGT GCACAGAGTC TTGG 3′,designated herein as SEQ ID NO:12. The primers were used to amplify PCRproducts from a C. familiaris mitogen activated PBMC cDNA library thatwas constructed in the Uni-ZAP® XR vector (available from StratageneCloning Systems, La Jolla, Calif.), using Stratagene's ZAP-cDNA®Synthesis Kit and the manufacturer's protocol. The mRNA was isolatedfrom C. familiaris peripheral blood mononuclear cells about 4 hoursafter they were activated by a polyclonal activating agent in culture.Four PCR products were produced that had the expected size range. ThePCR products were cloned and sequenced using standard techniques. Aportion of one of the four products was found to be somewhat homologouswith an IL-4 nucleic acid sequence reported in GenBank.

To identify a cDNA encoding a full-length canine IL-4 protein, the PCRproduct showing some homology with the IL-4 sequence reported in GenBankwas used to generate an about 549 base pair DNA fragment as follows. ThePCR product was labeled with ³²P and used as a probe to screen thecanine PBMC cDNA library. Hybridization was done at about 6×SSC,5×Denhardt's solution, 0.5% SDS, 100 μg/ml of ssDNA and 100 μg/ml oftRNA, at about 68° C., for about 36 hr. (the compositions of SSC andDenhardt's are described in Sambrook et al., ibid.). The filters werewashed 3 times, for about 30 minutes per wash, at about 55° C. in about2×SSC, 0.2% SDS, followed by a final wash of about 30 minutes in thesame buffer except using about 1×SSC. Positive clones were isolated andthe cDNA inserts were sequenced for both strands using vector flankingprimers and gene-specific internal primers. Sequence analysis wasperformed using the GAP program of GCG (available from the University ofWisconsin) using the alignment settings of: gap weight set at 50, lengthweight set at 3, and average match set at 10 for nucleic acid sequencecomparisons; and gap weight set at 12, length weight set at 4, andaverage match set at 2.912 for amino acid sequence comparisons.

A cDNA nucleic acid molecule was isolated, referred to herein asnCaIL-4₅₄₉, the coding strand of which was shown to have a nucleic acidsequence denoted herein as SEQ ID NO:1. The complement of SEQ ID NO:1 isrepresented herein by SEQ ID NO:3. Translation of SEQ ID NO:1 suggeststhat nucleic acid molecule nCaIL-4₅₄₉ encodes a full-length IL-4 proteinof about 132 amino acids, denoted herein as PCaIL-4₁₃₂, the amino acidsequence of which is presented in SEQ ID NO:2, assuming an open readingframe having an initiation codon spanning from nucleotide 43 throughnucleotide 45 of SEQ ID NO:1 and a stop codon spanning from nucleotide439 through nucleotide 441 of SEQ ID NO:1. The coding region encodingPCaIL-4₁₃₂ is presented herein as nCaIL-4₃₉₆, which has the nucleotidesequence SEQ ID NO:4 (the coding strand) and SEQ ID NO:5 (thecomplementary strand). A putative signal sequence coding region extendsfrom nucleotide 43 through nucleotide 114 of SEQ ID NO:1. The proposedmature protein (i.e., canine IL-4 protein from which the signal sequencehas been cleaved), denoted herein as PCaIL-4₁₀₈, contains about 108amino acids, extending from residue 25 through residue 132 of SEQ IDNO:2; PCaIL-4₁₀₈ amino acid sequence is represented herein as SEQ IDNO:20. The nucleic acid molecule encoding PCaIL-4₁₀₈ is denoted hereinas nCaIL-4₃₂₄, extending from nucleotide 115 through nucleotide 438 ofSEQ ID NO:1. nCaIL-4₃₂₄ has a coding sequence denoted SEQ ID NO:19 and acomplementary sequence denoted SEQ ID NO:21.

Comparison of nucleic acid sequence SEQ ID NO:1 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:1 showed the mosthomology, i.e., about 89.3% identity, with a feline IL-4 gene.Comparison of amino acid sequence SEQ ID NO:2 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:2 showed the most homology,i.e., about 82.6% identity, with a feline IL-4 protein. Sequenceanalysis was performed using the GCG GAP program as described above.

B. Expression of Recombinant Canine L-4 in E. coli and Mammalian Cells

i. E. coli expression

A recombinant molecule capable of expressing the mature form of canineIL-4, denoted herein as pGEX-nCaIL-4₃₂₇, was produced as follows. A340-nucleotide fragment was PCR amplified from nucleic acid moleculenCaIL-4₅₄₉ (having coding strand SEQ ID NO:1) using the following primersequences: positive strand 5′ TGAATTCGGA CATAACTTCA ATATTAC 3′ (SEQ IDNO:38) (EcoRI site in bold) and 5′ TCTCGAGATT CAGCTTCATG CCTGTA 3′ (SEQID NO39) (XhoI site in bold). The resulting 340-base pair fragment wasdigested with EcoRI and XhoI restriction enzymes (available from NewEngland Biolabs, Beverly, Mass.), according to the manufacture'sdirections, and gel-purified using standard techniques. The digested340-base pair fragment, now 327 base pairs, was then ligated intopGEX-6P-1 (available from Amersham Phartnacia, Piscataway, N.J.), whichhad been previously digested with EcoRI and XhoI and gel purified, toproduce recombinant molecule pGEX-nCaIL-4₃₂₇. Recombinant molecules ofpGEX produce the protein of interest as a glutathione s-transferase(GST) fusion protein. The recombinant molecule pGEX-nCaIL-4₃₂₇ wastransformed into DH5alpha cells (available from Life Technologies,Gaithersburg, Md.), a recombination deficient strain of E. coli, toproduce recombinant cell DH5-pGEX-nCaIL-4₃₂₇. The recombinant cells werescreened for presence of insert by PCR and confirmed by enzymerestriction analysis and nucleic acid sequencing, using standardtechniques. Several clonal recombinant molecules were transformed intoBL21 cells (available from Amersham Pharmacia, Piscataway, N.J.), aprotease deficient strain of E. coli, to produce recombinant cell BL21-pGEX-nCaIL-4₃₂₇. These recombinant cells were screened, and the clonewith the highest level of protein yield was selected for scaling up forlarger-scale protein production. The resultant recombinant protein isreferred to herein as E. coliPCaIL-4₁₀₉.

To produce and purify E. coliPCaIL-4₁₀₉, bacterial cultures ofrecombinant cell BL21; pGEX-nCaIL-4₃₂₇ were grown in shake flasks at 37°C. and induced with 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside),(available from Sigma Chemical Company, St. Louis, Mo.) when OD_(600nm)reached about 0.8 units. Growth was allowed to continue for about 4hours; then bacteria were harvested by centrifugation at 4000×g (timesgravity) for 20 minutes. The bacterial pellet was washed and resuspendedin phosphate buffered saline (PBS) (for recipe, see Sambrook et al,ibid.), then lysed by exposure to gaseous nitrogen pressure in a Parrpressure vessel (available from Parr Instrument Co., Moline, Ill.),according to the manufacturer's instructions. Cell debris was removed bycentrifugation at 10,000×g for 20 minutes. The IL-4-GST fusion proteinE. coliPCaIL-4₁₀₉ was purified from the supernatant by allowingincubation with glutathione-conjugated resin, removing unbound proteinsand then removing the GST tag with PRESCISSION® protease; all reagentswere available from Amersham Pharmacia and all were used according tothe manufacture's directions.

Concentration and purity of E. coliPCaIL-4₁₀₉ were estimated by BCAProtein Assay kit (available from Pierce, Rockford, Ill.) and SDS-PAGEfollowed by Coomassie staining, respectively. The purified materialexhibited a single band of approximately 14 kilodaltons (kD) byCoomassie stained SDS-PAGE.

ii. CHO cell expression

A recombinant molecule denoted herein as pCMV-nCaIL-4₃₉₉, capable ofexpressing a full length form of canine IL-4 (including signal sequence)was produced as follows. A 422-nucleotide fragment was PCR amplifiedfrom nucleic acid molecule nCaIL-4₅₄₉ using the following primers: 5′CCCAAGCTTA TGGGTCTCACC TCCCAAC (HindIII site in bold), denoted SEQ IDNO:40, and 3′ CCTCGAGATT CAGCTTTCAA TGCCTGTA (XhoI site in bold),denoted SEQ ID NO:127. The 422-base pair PCR product was digested withthe restriction endonucleases HindIII and XhoI, both available from NewEngland Biolabs. The resulting 399-base pair product encodingfull-length canine IL-4 was gel purified using standard techniques andligated into the cytomegalovirus (CMV) immediate-early transcriptioncontrol region of the pCMV-Int A plasmid vector that had been digestedwith HindIII and XhoI (available from New England Biolabs), and gelpurified, to produce the recombinant molecule pCMV-nCaIL-4₃₉₉. ThepCMV-Int A plasmid vector was generated as referenced by J. E. Osorio etal., 1999, Vaccine 17, 1109-1116. Briefly, vector pRc/RSV, (availablefrom Invitrogen Corp., San Diego, Calif.) was cleaved with restrictionenzyme PvuII (available from New England Biolabs), and the 2963-basepair PvuII fragment was gel purified. The fragment was self-ligated toform the vector pRc/RSV(Pvu), which contains a Rous Sarcoma Virus (RSV)long terminal repeat, a multiple cloning site, a bovine growth hormonepolyadenylation sequence, a bacterial origin of replication, and anampicillin resistance gene. Vector pRc/RSV(Pvu) was restriction enzymedigested using HindIII and NruI. A HindIII/SspI fragment containing theHCMV intermediate early promoter and first intron (i.e. intron A) wasligated into the digested pRc/RSV(Pvu) vector to produce the vectorpCMV-Int A.

Stable expression of CaIL-4 in mammalian cells was carried out bytransfecting the recombinant molecule pCMV-nCaIL-4₃₉₉ into ChineseHamster Ovary cells, (CHO, available from ATCC) as follows. Six-wellpolystyrene tissue culture plates (available from Coming Costar, Acton,Mass.) were seeded with approximately 5×10⁵ cells/well in 2 milliliter(ml) cell culture media, consisting of Minimal Essential Media (MEM)supplemented with 100 mM L-glutamine, 100 mM gentamicin, and 10% fetalbovine serum (FBS), (all available from Life Technologies). Cells weregrown to about 80% confluence (for about 18 hours) before transfection.The recombinant molecules to be transfected were purified using thePlasmid Midi Kit (available from Qiagen, Valencia, Calif.) and usedaccording to the manufacture's instructions. The recombinant moleculepCMV-nCaIL-4₃₉₉ was linearized using the restriction enzyme PvuI(available from New England Biolabs). The plasmid pcDNA3, (availablefrom Invitrogen), which contains the neomycin resistance gene, waslinearized using the restriction enzyme EcoRI. Approximately 2 μg ofpCMV-nCaIL-4₃₉₉ was mixed with about 2 ng of linearized pcDNA3 in about100 μl OPTIMEM™ media, available from Life Technologies. About 10 μlLipofectamine, (available from Life Technologies) was mixed with 100 μlOPTIMEM. The nucleic acid molecule-containing mixture was then added tothe Lipofectamine mixture and incubated at room temperature for about 45minutes. After incubation, about 0.8 ml OPTIMEM was added, and themixture was overlaid onto the CHO cells which had been previously rinsedwith OPTIMEM. Cells were incubated for about 5 hours at 37° C. 5% CO₂,95% relative humidity. Approximately 1 ml of cell culture media asdescribed previously, with 20% FBS, was added and the cells wereincubated overnight. The media was changed at 24 hours, and at 72 hourspost transfection, the cells were split 1:4 and put into fresh cellculture media containing about 500 μg/ml geneticin (G418, available fromLife Technologies). The media was changed every 3-5 days. After severalweeks, G418 resistant colonies were trypsinized using sterile filterpapers, 5-6 mm in diameter that were soaked in trypsin, which were thenplaced over individual well of 24 well plates that contained separatedwidely spaced colonies of CHO cells. After 3 days, the papers wereremoved. The resulting recombinant cells are referred to herein asCHO-pCMV-nCaIL-4₃₉₉. The recombinant cells were then expanded and testedfor the presence of nIL-4₃₉₉ RNA by RT-PCR and tested for the presenceof PCaIL-4₁₃₃ protein by Western blot analysis. Westerns were developedwith rabbit anti-E. coliPCaIL-4₁₀₉ serum and 607.1 monoclonal antibody,a monoclonal antibody that selectively binds to E. coliPCaIL-4₁₀₉protein. See Example 1C for a description of how these antibodies wereproduced.

C. Monoclonal and Polyclonal Antibodies to Recombinant Canine IL-4 (i.e.Anti-canine IL-4 Antibodies)

The following describes the development of monoclonal and polyclonalantibodies that selectively bind to E. coliPCaIL-4₁₀₉.

Female Balb/C mice, 6-8 weeks old, were injected subcutaneously, atabout 4 sites, with a total of 25 μg E. coliPCaIL-4₁₀₉ (produced asdescribed in Example 1B) in Freund's Complete Adjuvant (day 0). Fourteendays later, the mice received an intraperitoneal boost of 25 μg E.coliPCaIL-4₁₀₉ in Freund's Incomplete Adjuvant (day 14). Fourteen dayslater, serum was tested for antibody titer to E. coliPCaIL-4₁₀₉ by ELISA(day 28). Three days prior to fusion, mice were boosted intravenouslywith 20 μg E. coliPCaIL-4₁₀₉ in PBS (day 35). Splenocytes were harvestedfrom mice demonstrating the highest serum titer by ELISA and depleted ofCD4+ and CD8′ cells. This depletion was achieved by incubation of thesplenocytes with biotinylated rat anti-mouse CD4 and anti-mouse CD8monoclonal antibodies, available from PharMingen, San Diego, Calif.Antibody-labeled cells were then removed by incubation with M-280streptavidin coated magnetic beads, available from Dynal, Oslo, Norway.Depleted splenocytes were fused to SP2/0 cells (valuable from ATCC)using 50% polyethylene glycol in unsupplemented Iscove's ModifiedDulbecco's Media (IMDM), following established protocols; see, forexample, Harlow E., and Lane D., eds., 1995, Antibodies, A LaboratoryManual, Monoclonal Antibodies, Cold Spring Harbor Laboratories; Harlowet al, ibid., is incorporated by reference herein in its entirety. Fusedcells were plated in 96-well plates using IMDM cell culture media,(available from Life Technologies, Inc., Rockville, Md.), which wassupplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodiumpyruvate, 1×nonessential amino acids, 1×MEM amino acids, 0.05 mg/mlgentamicin, and 0.5 mM β-mercaptoethanol (all reagents available fromLife Technologies). Additionally, 100 μM hypoxanthine, 0.4 μMaminopterin, and 16 μM thymidine, all available from Sigma ChemicalCorporation, St Louis, Mo., were added.

After about 7 days, wells positive for hybirdoma growth were screened byELISA to E. coliPCaIL-4₁₀₉. Immulon II 96-well plates (available fromVWR, Denver, Colo.) were coated, overnight, with 100 ng/ml E.coliPCaIL-4₁₀₉ in 0.1 M carbonate/bicarbonate buffer, Ph 9.6. Afterblocking the wells with 20% FBS in Tris buffered saline (TBS), culturesupernatants were allowed to bind. Presence of anti-E. coliPCaIL-4₁₀₉mouse antibody was detected with polyclonal goat anti-mouse IgGconjugated to horseradish peroxidase, (available from KPL, Gaithersburg,Md.), and color developed with 3,3′,5,5′ -tetramethylbenzidinedihydrochloride (TMB), available from Pierce, Rockford, Ill. Specificityof the ELISA reactivity was verified by Western blot analysis to E.coliPCaIL-4₁₀₉, developed with polyclonal goat anti-mouse IgG conjugatedto alkaline phosphatase and nitro-bluetetrazolium/5-bromo-4-chloro-3′-indolyphophate p-toluidine saltsubstrate (NBT/BCIP, available from Sigma). Western blots exhibited asingle band of approximately 14 kD. Immunoglobulin isotype of themonoclonal antibodies was determined using IsoStrips, available fromBoehringer Mannheim, Indianapolis, Ind. Twenty-three monoclonalantibodies were generated to E. coliPCaIL-4₁₀₉, 22 of which were of theIgM isotype and one of which was IgG1, and is referred to herein as607.1.

Polyclonal rabbit serum was produced by repeated immunization (over a 10month period) of a male, New Zealand White rabbit 12-16 months old.Initial immunization was 50 μg E. coliPCaIL-4₁₀₉ (prepared as describedin Example 1bi) in Freund's Complete Adjuvant, at several sitessubcutaneously and intradermally. One month later, and at one monthintervals thereafter, the rabbit was boosted intradermally with 50 ug E.coliPCaIL-4₁₀₉ in Freund's Incomplete Adjuvant. Serum was collectedbi-weekly and titers monitored by ELISA and Western blot to E.coliPCaIL-4₁₀₉. Serum that selectively bound to E. coliPCaIL-4₁₀₉protein is referred to as anti-E. coliPCaIL-4₁₀₉ serum.

D. Bioactivity of Mammalian-Expressed Canine IL-4

The following describes a bioassay to detect the expression of canineIL-4 protein expressed in the supernatants from CHO-pCMV-nCaIL-4₃₉₉recombinant cells by screening for production of CD23.

About 100 μl Ramos cells, available from ATCC, at a concentration ofabout 3.5×10³ cells/ml were seeded into 96-well flat bottom plates,available from Becton Dickinson, Franklin Lakes, N.J.). These cells weregrown in RPMI media supplemented with 100 mM L-glutamine, gentamicin,and 10% FBS (called TCM). The Ramos cells were then treated in 5% CO₂for 37° C. for approximately 48 h. with one of the following:

Group Treatment 1 TCM 2 CHO-pCMV (a transfectant cell line containingthe empty pCMV vector) supernatant (1:4 final dilution in TCM) 3CHO-pCMV-nCaIL-4₃₉₉ supernatant (1:10 final dilution in TCM)

Triplicate samples for each treatment group were pooled for staining tolook for increased expression of CD23 (one of the reported effects ofIL-4). Briefly, 1×10⁵ cells from each treatment group were incubated inphosphate buffered saline (PBS) containing 30% FBS for 15-30 min on ice.The cells were collected and incubated with the following:

Condition Primary Incubation Secondary Incubation A PBS Goat anti mousePE B Mouse anti human CD23 Goat anti mouse PE

Mouse anti-human CD23 monoclonal antibody, available from Pharmingen,was used at about 10 μg/ml. Goat (Fab'2) anti mouse IgG PE, availablefrom Southern Biotechnologies was used at about 2.5 μg/ml. Thesereagents were diluted in PBS with 5% FBS . Primary incubations wereperformed for 30-60 minutes on ice, and secondary incubations wereperformed for 20-30 min on ice. Three washes of PBS/5% FBS wereperformed in between each incubation. Cells were then analyzed on a flowcytometer (e.g., MoFlow Desk Top System, available from Cytomation, Ft.Collins, Colo.) with the fluorescein gate set at 10¹. The results areshown in Table 2.

TABLE 2 Incubation of CD23 on Ramos cells post-treatment withsupernatants from CHO-pCMV-nCaIL-4₃₉₉ . Treatment Conditions % positive1 A 0 B 1 2 A 8 B 1 3 A 3 B 99

Table 2 shows that the canine II-4 expressed by the CHO transfectantCHO-pCMV-nCaIL-4₃₉₉is biologically active, demonstrated by its abilityto induce expression of CD23 in Ramos cells.

E. Bioactivity of E. coli-expressed Canine IL-4

The following describes a bioassay to detect the expression of canineIL-4 by stimulating the proliferation of TF-1 cells.

TF-1 cells (a human erytliroleukaemia cell line, available from R&DSystems, Minneapolis, Minn.), were grown and maintained in TCM-TF-1medium (RPMI-1640 media supplemented with 2 mM L-glutamine, 5 μg/mlgentamicin, 5% FBS and 2 ng/ml recombinant human GM-CSF (rhuGM-CSF,available from R&D Systems)) in 5% CO₂ at 37° C.

For assay, TF-1 cells were extensively washed to remove rhuGM-CSF, thenadded at approximately 1×10⁴ cells per well to 96-well flat bottomplates. Refolded and HPLC-purified E. coli-expressed PCaIL-4₁₀₉,produced as described in Example 1Bi, was diluted to the appropriateconcentration in TCM-TF-1 without rhuGM-CSF and filter sterilized. Cellsand E. coli-expressed PCaIL-4₁₀₉ were incubated for 48 hours in 5% CO₂at 37° C., then pulsed with 1 μCi/well tritiated thymidine (availablefrom ICN Pharmaceuticals, Irvine, Calif.), and incubated for anadditional 18 hours. Contents of the wells were harvested onto glassfiber filters and counted in a Wallace Trilux 1450 scintillation counter(available from Wallac Inc., Gaithersburg, Md.). The results are shownin Table 3.

TABLE 3 Stimulation of proliferation of TF-1 cells with E.coli-expressed PCaIL-4₁₀₉ Concentration E. coli PCaIL-4₁₀₉ (ng/ml)Counts per minute 1000 33,216 500 26,297 250 27,283 125 23,804 62.526,225 31.3 19,803 15.6 9,818 7.8 6,475 0 165

Table 3 shows that canine IL-4 expressed by E. coli is biologicallyactive, as demonstrated by its ability to stimulate proliferation ofTF-1 cells.

Example 2

This example describes the isolation and sequencing of certain canineFlt-3 ligand and feline Flt-3 nucleic acid molecules and proteins of thepresent invention. The example also describes expression of a canineFlt-3 ligand protein of the present invention in CHO cells, as well asdetection of the expressed canine Flt-3 ligand protein.

A. Canine Flt-3 Ligand Nucleic Acid Molecules and Proteins.

i. This example describes the isolation and sequencing of certain canineFlt-3 ligand nucleic acid molecules and proteins of the presentinvention.

A canine Flt-3 ligand nucleic acid molecule was produced as follows. Apair of primers was initially used to amplify DNA from the C. familiarismitogen activated PBMC cDNA library described above in Example 1. Aforward primer referred to as FLT3F1, having the nucleic acid sequence5′ CTGGCGCCAG CCTGGAGCCC 3′, designated herein as SEQ ID NO:13 was usedin combination with a reverse primer referred to herein as FLT3B1,having the nucleic acid sequence 5′ GGGAGATGTT GGTCTGGACG 3′, referredto herein as SEQ ID NO:14 to amplify Flt-3 ligand DNA from the cDNAlibrary by polymerase chain reaction (PCR). The primers were designedusing conserved regions of IL-4 cDNA sequences from other species in thepublic databases corresponding to the positions shown below:

Database Accession number Nucleotides Animal gb U04806 102-121 human gbL23636 41-60 mouse gb U04806  77-458 human gb L23636 419-400 mouse

A 360-base pair (bp) PCR product was generated in the above reactionthat was purified, radiolabeled and used as a probe to screen the cDNAlibrary. Hybridization was performed in 6×SSC, 5×Denhardt's solution,0.5% SDS, 100 μg/ml ssDNA and 100 μg/ml of tRNA, at 68° C., for about 36hr. The filters were washed 3 times, for about 30 minutes per wash, at55° C. in 2×SSC, 0.1% SDS, followed by a final wash in 0.25×SSC, forabout 30 minutes, at 55° C. Several positive phage clones wereidentified and shown to produce PCR products when used as templates incombination with the FLT3F1 and FLT3B1 primers. The DNA inserts in thephage clones were sequenced using standard techniques and failed toyield any clones containing DNA inserts having a portion homologous topublished Flt-3 ligand sequences. The 360-bp PCR fragment generatedabove was then cloned into the vector pcDNA 2.1 (available fromInvitrogen Corp., San Diego, Calif.). Several independent colonies weregenerated and the sequences of their inserts determined. One clone wasidentified that which contained insert sequence having a portion thatwas somewhat homologous to published human or murine Flt-3 ligandsequence.

Two canine Flt-3 ligand-specific primers were then designed using thenucleic acid sequence obtained using the 360-bp PCR product describedabove.

Primer Sequence SEQ ID NO DFLB1 5′ GACCAGGCGCCAGAACGC 3′ SEQ ID NO: 15DFLF1 5′ CGGTCACCATCCGCAAGC 3′ SEQ ID NO: 16

The 5′ region of a Flt-3 ligand nucleic acid molecule was PCR amplifiedfrom the cDNA library using the DFLB1 primer in combination with the 5′T3 vector primer from the Uni-ZAP® XR vector (available fromStratagene). The 3′ region of a Flt-3 ligand nucleic acid molecule wasPCR amplified from the cDNA library using the DFLF1 in combination withthe 3′ T7 primer from the Uni-ZAP® XR vector (available fromStrategene). A 855-bp PCR product was obtained representing the 5′region of a Flt-3 ligand nucleic acid molecule. A 265-bp PCR product wasobtained representing the 3′ region of a Flt-3 ligand nucleic acidmolecule. Both the 855-bp PCR product and 265-bp PCR product were clonedand sequenced using standard methods. Additional canine Flt-3ligand-specific primers were designed using the nucleic acid sequenceobtained from the sequence of the 855-bp PCR product and 265-bp PCRproducts.

Primer Sequence SEQ ID NO DFLB2 5′ TGGCAAGGCAGTGGCCTC 3′ SEQ ID NO: 17DFLF2 5′ GCCGAGATGATAGTGCTGGC 3′ SEQ ID NO: 18

A 546-bp PCR product was generated using the primer DFLF2 in combinationwith the primer DFLB2 to amplify a PCR product from the cDNA library.The 546-bp PCR product was then purified, radiolabelled and used as aprobe to screen the cDNA library. Hybridization was performed in 6×SSC,5×Denhardt's solution, 0.5% SDS, 100 μg/ml of ssDNA, and 100 μg/ml oftRNA, at 68° C., for about 36 hr. The filters were washed in 1.25×SSC,for about 30 minutes, at 55° C. Four cDNA clones encoding full-lengthcanine Flt-3 ligand were isolated. Nucleic acid sequence was obtainedusing standard techniques.

A Flt-3 ligand clone was isolated, referred to herein as nCaFlt3L₁₀₁₃,the coding strand of which was shown to have a nucleic acid sequencedenoted herein as SEQ ID NO:6. The complement of SEQ ID NO:6 isrepresented herein by SEQ ID NO:8. Translation of SEQ ID NO:6 suggeststhat nucleic acid molecule nCaFlt3L₁₀₁₃ encodes a full-length Flt-3ligand protein of about 294 amino acids, denoted herein as PCaFlt3L₂₉₄,the amino acid sequence of which is presented in SEQ ID NO:7, assumingan open reading frame having an initiation codon spanning fromnucleotide 35 through nucleotide 37 of SEQ ID NO:6 and a stop codonspanning from nucleotide 917 through nucleotide 919 of SEQ ID NO:6. Thecoding region encoding PCaFlt3L₂₉₄ is presented herein as nCaFlt3L₈₈₂,which has the nucleotide sequence SEQ ID NO:9 (the coding strand) andSEQ ID NO:10 (the complementary strand). A putative signal sequencecoding region extends from nucleotide 35 through nucleotide 112 of SEQID NO:6. The proposed mature protein (i.e., canine Flt-3 ligand proteinfrom which the signal sequence has been cleaved), denoted herein asPCaFlt3L₂₆₈ (SEQ ID NO:23), contains about 268 amino acids, extendingfrom residue 27 through residue 294 of SEQ ID NO:7. The nucleic acidmolecule encoding PCaFlt3L₂₆₈ is denoted herein as nCaFlt3L₈₀₄,extending from nucleotide 113 through nucleotide 916 of SEQ ID NO:6.nCaFlt3L₈₀₄ has a coding sequence denoted SEQ ID NO:22 and acomplementary sequence denoted SEQ ID NO:24.

Below is a description of the identification of alternatively splicedCanis Flt3 ligand transcripts. Besides cDNA clones such as nucleic acidmolecule nCaFlt3L₁₀₁₃ encoding the full-length canine Flt3 ligandprotein, two splice variants of canine Flt3 ligand cDNA clones were alsoisolated, using the same hybridization conditions as mentionedpreviously in this Example. One such variant (Clone 1), denoted hereinas nCaFlt3L₉₈₅, has a coding strand the nucleic acid sequence of whichis represented as SEQ ID NO:25. The complement of SEQ ID NO:25 isrepresented herein by SEQ ID NO:27. Translation of SEQ ID NO:25 suggeststhat nucleic acid molecule nCaFlt3L₉₈₅ encodes a Flt-3 ligand protein of276 amino acids, denoted herein as PCaFlt3L₂₇₆, the amino acid sequenceof which is represented by SEQ ID NO:26, assuming an open reading framehaving an initiation codon spanning from nucleotide 74 throughnucleotide 76 of SEQ ID NO:25 and a stop codon spanning from nucleotide902 through nucleotide 904 of SEQ ID NO:25. The coding region encodingPCaFlt3L₂₇₆ is represented herein as nCaFlt3L828, which has thenucleotide sequence SEQ ID NO:28 (the coding strand) and SEQ ID NO:29(the complementary strand). Alignment of nucleic acid moleculesnCaFlt3L₈₈₂ and nCaFlt3L₈₂₈ indicates that the nucleic acid moleculesare identical except for a deletion in nCaFlt3L₈₂₈, which spans fromnucleotide 343 through nucleotide 396 of nCaFlt3L₈₈₂. Accordingly,nCaFlt3L₈₂₈ encodes 18 fewer amino acids than nCaFlt3L₈₈₂. The deletionin PCaFlt3L₂₇₆, which spans from residue 115 through residue 132 ofPCaFlt3L₂₉₄, occurs between helix III and helix IV of the canine Flt3ligand protein inferred from alignment with the human and mouse Flt3ligand protein (Lyman et al., Cell, vol. 75, pp. 1157-1167, 1993; Hannumet al., Nature, vol. 368, pp. 643-648, 1994; Lyamn et al., Blood, vol.83, pp. 2795-2801, 1994). In addition, the alignment shows that thereare 39 more nucleotides in the 5′ untranslated region of nucleic acidmolecule nCaFlt3L₉₈₅ (nucleotides 1 to 39) than nucleic acid moleculenCaFlt3L₁₀₁₃ and there are 2 more nucleotides in the 3′ untranslatedregion of nucleic acid molecule n CaFlt3 L₉₈₅ (nucleotides 922 to 923)than nucleic acid molecule nCaFlt3L₁₀₁₃. The remaining sequences betweennCaFlt3L₉₈₅ and nCaFlt3L₁₀₁₃ are identical. A putative mature form ofnCaFlt3L₉₈₅ (without the signal sequence) is predicted. The putativesignal sequence coding region extends from nucleotide 74 to nucleotide151 of SEQ ID NO:25. The proposed mature protein, denoted herein asPCaFlt3L₂₅₀, represented by SEQ ID NO:31, contains about 250 aminoacids, extending from residue 27 through residue 276 of SEQ ID NO:26.The nucleic acid molecule encoding PCaFlt3L₂₅₀, extending fromnucleotide 152 through nucleotide 901 of SEQ ID NO:6, denoted herein asnCaFlt3L₇₅₀, is represented by SEQ ID NO:30 (the coding strand) and SEQID NO:32 (the complement strand).

A second variant (Clone 19) is represented by nucleic acid moleculenCaFlt3L₁₀₁₉, the coding strand of which is denoted herein as SEQ IDNO:33. The component of SEQ ID NO:33 is denoted herein as SEQ ID NO:35.Translation of SEQ ID NO:33 suggests that nCaFlt3L₁₀₁₉ encodes a Flt-3ligand protein of 31 amino acids, PCaFlt3L₃₁, denoted SEQ ID NO:34,assuming an initiation codon spanning from nucleotide 74 throughnucleotide 76 and a stop codon spanning nucleotide 167 throughnucleotide 169 of SEQ ID NO:33. The coding region encoding PCaFlt3L₃₁ isrepresented herein as nCaFlt3L₉₃, which has the nucleotide sequence SEQID NO:36 (the coding strand) and SEQ ID NO:37 (the complementarystrand). Alignment of nucleic acid molecules nCaFlt3L₉₈₅ andnCaFlt3L₁₀₁₉ indicates the presence of an insertion of 91 nucleotides innCaFlt3L₁₀₁₉. The insertion spans nucleotide 107 through nucleotide 198of nCaFlt3L₁₀₁₉. A stop codon is found in this insertion in frame withthe predicted initiation codon, which span nucleotide 74 throughnucleotide 76 of SEQ ID NO:6. Since this insertion (with an inframe stopcodon) occurs in or close to the signal peptide, it is likely thatnucleic acid molecule nCaFlt3L₁₀₁₉ encodes a nonfunctional Flt-3 ligandprotein.

Comparison of nucleic acid sequence SEQ ID NO:6 with nucleic acidsequences reported in GenBank indicates that SEQ ID NO:6 showed the mosthomology, i.e., about 69.8% identity, with a human Flt-3 ligand gene.Comparison of amino acid sequence SEQ ID NO:7 with amino acid sequencesreported in GenBank indicates that SEQ ID NO:7 showed the most homology,i.e. about 71% identity, with a human Flt-3 ligand protein. Sequenceanalysis was performed with DNAsis™ using the alignment settings of: gappenalty set at 5; number of top diagonals set at 5; fixed gap penaltyset at 10; K-tuple set at 2; window size set at 5 and floating gappenalty set at 10.

ii. This example describes the production of a recombinant moleculeencoding a full length canine Flt-3 ligand protein and expression ofthat protein by a recombinant cell of the present invention.

A recombinant molecule, denoted herein as pCMV-nCaFlt3L₈₈₂ and capableof expressing a full length form of Flt-3 ligand, was produced asfollows. Nucleic acid molecule nCaFlt3L₈₈₂ was digested with therestriction endonucleases EcoRI and XbaI, gel purified and ligated intopCMV-Int A (prepared by methods described in Example 1) to producerecombinant molecule pCMV-nCaFlt3L₈₈₂. Insert size and identity wereconfirmed by restriction digestion, PCR, and sequencing analyses.

Stable transfectants expressing the recombinant moleculepCMV-nCaFlt3L₈₈₂ were established in Chinese Hamster Ovary cells (CHO,available from ATCC) as follows. Briefly, six-well polystyrene tissueculture plates were seeded with approximately 4×10⁵ cells per well in 2ml of MEM (available from Life Technologies, Gaithersburg, Md.)supplemented with 100 mM L-glutamine, gentamicin, and 10% FBS (TCM).Cells were grown to about 80% confluence (about 18 hr). The recombinantmolecule to be transfected was prepared using the Qiagen Endotoxin-FreePlasmid Maxi Kit as per the manufacturer's instructions. The recombinantmolecule was linearized with the restriction enzyme PvuI, extracted withphenol, and precipitated with isopropanol. The plasmid pcDNA 3,available from Invitrogen, which contains the neomycin resistance gene,was linearized with the restriction enzyme EcoRI. Approximately 1 μg ofrecombinant plasmid DNA and 100 ng of pcDNA3 were mixed with about 100μl OptiMEM medium, available from Life Technologies. About 10 μlLipofectamine (available from Life Technologies) was mixed with 100 μlOptiMEM. The DNA-containing mixture was then added to the Lipofectaminemixture and incubated at room temperature for about 30 min. Afterincubation, about 800 μl of OptiMEM was added, and the entire mixturewas overlaid onto the CHO cells that had been rinsed with OptiMEM. Cellswere incubated for 6 hours at 37° C., 5% CO₂, 95% relative humidity.Approximately 1 ml of TCM with 20% FBS was added, and the cells wereincubated overnight. The media was changed after about 24 hr. About 72hr post transfection, the cells were split 1:4 and put into selectionTCM containing 500 μg/ml Geneticin (G418), available from LifeTechnologies. The medium was changed every 3-5 days. After severalweeks, G418-resistant colonies were trypsinized, and the cells wereplated into 24 well plates. The resulting recombinant cells are referredto herein as CHO-pCMV-nCaFlt3L₈₈₂. The recombinant cells were thenexpanded for testing.

iii. The following describes the detection of expression of a canineFlt-3 ligand protein of the present invention by CHO-pCMV-nCaFlt3L₈₈₂, arecombinant cell of the present invention.

Recombinant cells CHO-pCMV-nCaFlt3L₈₈₂, produced as described in Example2, part (B)(ii) above, were tested for surface expression of canineFlt-3 ligand using a cross-reactive goat anti-human Flt-3 ligandpolyclonal antibody as follows. Briefly, 1×10⁵ CHO-pCMV-nCaFlt3L₈₈₂cells or CHO-pCMV cells (i.e., cells transfected with an empty vector asdescribed in Example 1) were incubated in phosphate buffered saline(PBS) containing 30% fetal bovine serum (FBS) for about 30 min on ice.The cells were then spun down and treated with the following:

Condition Primary Incubation Secondary Incubation 1 PBS Rabbit (Fab′2)anti sheep (H + L) FITC 2 Goat anti-human Flt3 ligand Rabbit (Fab′2)anti sheep (H + L) FITCGoat anti-human Flt3 ligand, available from R and D Systems,Minneapolis, Minn. was used at about 20 μg/ml. Rabbit (Fab′2) anti sheep(H+L) FTTC, available from Southern Biotechnology Associates, Inc., wasused at about 10 μg/ml. These reagents were diluted in PBS/5%FBS. Allincubations were in 50 μl for about 1 hr on ice with 2 washes of PBS/5%FBS in between each incubation. Cells were then analyzed on a flowcytometer (e.g., MoFlow Desk Top System, available from Cytomation, Ft.Collins, Colo.) with the fluorescein gate set at 10¹. The results areshown be low in Table 4.

TABLE 4 Expression of canine Flt3 ligand on CHO transfectants. %positive Cells Condition 1 Condition 2 CHO-pCMV 1 1 CHO-pCMV nCaFlt3L₈₈₂2 48 CHO-pCMV nCaFlt3L₈₈₂ 1 20

Table 4 shows that canine Flt3 ligand is expressed on the surface of theCHO transfectants.

B. Feline Flt-3 Ligand Nucleic Acid Molecules and Proteins.

This example describes the production of certain feline Flt-3 ligandnucleic acid molecules and proteins of the present invention.

A nucleic acid molecule encoding a feline Flt 3 ligand was isolated froma feline PBMC cDNA library as follows. A Felis catus mitogen activatedPBMC cDNA library was constructed in the Uni-Zap-R XR™ vector, availablefrom Stratagene, La Jolla, Calif., using Stratagene's Zap-cDNA-R™Synthesis Kit and the manufacturer's protocol using mRNA isolated fromF. catus peripheral blood mononuclear cells about 4 hours after theywere activated by a polyclonal activating agent in culture. PCRamplification to isolate a feline Flt 3 ligand nucleic acid molecule wasconducted according to the following set of steps: one initialdenaturation step at 95° C. for 3 minutes; then 35 cycles of thefollowing: 94° C. for 30 seconds, 53.8° C. for 30 seconds, and 72° C.for 105 seconds; then one final extension step at 72° C. for 8 minutes.A 395-nucleotide cDNA fragment containing the 5′ end of feline Flt3ligand coding region, denoted c eFlt3L₃₉₅,was amplified from the felinePMBC cDNA library using the following primers: vector primer T3 havingnucleic acid sequence 5′ AATTAACCCT CACTAAAGGG 3, (SEQ ID NO:142)(available from Stratagene) and the antisense primer having SEQ IDNO:14, described in Example 2A. The nucleic ac id sequence of the codingstrand of nFeFlt3L₃₉₅ is denoted SEQ ID NO:41. A 793-nucleotide cDNAfragment containing the3′ end of feline Flt3 ligand coding region,denoted nFeFlt3L₇₉₃, was isolated using sense primer 2 having thenucleic acid sequence 5′ CACAGYCCCA TCTCCTCC 3, (where Y was either T orC) denoted herein as SEQ ID NO:151, in conjunction with vector primer T7having the nucleic acid sequence 5′ GTAATACGAC TCACTATAGG GC 3′ (SEQ IDNO:152). The nuclei c acid sequence of the coding strand of nFeFlt3L₇₉₃is denoted SEQ ID NO:42. Nucleic acid molecules feFlt3L₃₉₅ andnFeFlt3L₇₉₃ overlap by 246 nucleotides and form a composite sequenceencoding a Flt3 ligand protein that is similar in length to that ofPCaFlt3L₂₉₄. This composite feline Flt3 ligand cDNA is referred toherein as nFeFlt3L₉₄₂, the coding strand of which was shown to havenucleic acid sequence SEQ ID NO:43. The reverse complement of SEQ IDNO:43 is referred to herein as SEQ ID NO:45. Translation of SEQ ID NO:43suggests that nucleic acid molecule nFeFlt3L₉₄₂ encodes a Flt3 ligandprotein of 291 amino acids, denoted herein as PeFlt3L₂₉₁, the amino acidsequence of which is presented in SEQ ID NO:44, assuming an open readingframe having an initiation codon spanning from nucleotide 31 throughnucleotide 33 of SEQ ID NO:43 and a stop codon spanning from nucleotide904 through nucleotide 906 of SEQ ID NO:43. The coding region encodingPFeFlt3L₂₉₁, not including the termination codon, is presented herein asnFeFlt3L₈₇₃, which has the nucleotide sequence SEQ ID NO:46 (the codingstrand) and SEQ ID NO:47 (the complementary strand). A putative signalsequence coding region extends from nucleotide 31 to nucleotide 108 ofSEQ ID NO:43. The proposed mature protein, denoted herein asPFeFlt3L₂₆₅, denoted SEQ ID NO:49, contains about 265 amino acids,extending from residue 27 though residue 291 of SEQ ID NO:44. Thenucleic acid molecule encoding PFeFlt3L₂₆₅ is denoted herein asnFeFlt3L₇₉₅, (SEQ ID NO:48) extending from nucleotide 109 throughnucleotide 903 of SEQ ID NO:43. SEQ ID NO:48 has a complementary stranddenoted SEQ ID NO:50.

Sequence alignment indicates that nucleic acid sequence SEQ ID NO:43shares the highest (67.8%) identity with the nucleic acid sequence ofhuman Flt-3 ligand (GenBank accession numbers U04806 and U03858). Aminoacid sequence SEQ ID NO:44 shares the highest (70.2%) identity withhuman Flt-3 ligand protein (GenBank accession numbers U04806 andU03858).

Example 3

This examples describes the isolation and sequencing of certain canineCD40 and feline CD40 nucleic acid molecules and proteins of the presentinvention.

A. Canine CD40 Nucleic Acid Molecules and Proteins

This example describes the production of certain canine CD40 nucleicacid molecules and proteins of the present invention.

A canine CD40 nucleic acid molecule of the present invention wasproduced by PCR amplification as follows. A 321-nucleotide canine CD40nucleic acid molecule, denoted nCaCD40₃₂₁, was amplified from a caninePBMC cDNA library, prepared as described in Example 1, using twodegenerate oligonucleotide primers designed in accordance with conservedregions of human, bovine, rabbit, and mouse CD40 gene sequencesavailable in GenBank; sense primer, 5′ TGCCCRSTCG GCTTCTTCTC C 3′,denoted herein as SEQ ID NO:128; and antisense primer, 5′ CGACTCTCTTTRCCRTCCTC CTG 3′, denoted herein as SEQ ID NO:129, where R was either Aor G and S was either G or C. PCR conditions were as follows: oneinitial denaturation step at 95° C. for 3 minutes; then 35 cycles of thefollowing: 94° C. for 30 seconds, then 53° C. for 30 seconds, then 72°C. for 105 seconds; followed by one final extension at 72° C. for 5minutes. The resulting PCR product, i.e., nCaCD40₃₂₁, with a codingstrand represented by SEQ ID NO:51, was radiolabeled using standardtechniques and used to screen the canine PBMC cDNA library, under thefollowing hybridization conditions: hybridized in 6×SSC, 5×Denhardt'ssolution, 0.5% SDS, 100 μg/ml single stranded DNA, 100 μg/ml tRNA for 36hours at 68° C., followed by a wash of 0.1% SDS, 1×SSC at 55° C. for 60minutes. A clone (Clone 18B) containing a 1425-nucleotide canine CD40nucleic acid molecule, denoted nCaCD40₁₄₂₅, was obtained. The nucleicacid sequence of the coding strand of nCaCD40₁₄₂₅ is represented as SEQID NO:52. The reverse complement of SEQ ID NO:52 is referred to hereinas SEQ ID NO:54. Translation of SEQ ID NO:52 suggests that nucleic acidmolecule nCaCD40₁₄₂₅ encodes a canine CD40 protein of 274 amino acids,denoted herein as PCaCD40₂₇₄, the amino acid sequence of which ispresented in SEQ ID NO:53, assuming an open reading frame having aninitiation codon spanning from nucleotide 196 through nucleotide 198 ofSEQ ID NO:52 and a stop codon spanning from nucleotide 1018 throughnucleotide 1020 of SEQ ID NO:52. The coding region encoding PCaCD40₂₇₄,not including the termination codon, is presented herein as nCaCD40₈₂₂,which has the nucleotide sequence SEQ ID NO:55 (the coding stand) andSEQ ID NO:56 (the complementary strand).

A putative signal sequence coding region extends from nucleotide 196through nucleotide 252 of SEQ ID NO:52. The proposed mature protein,denoted herein as PCaCD40₂₅₅, represented by SEQ ID NO:58, containsabout 255 amino acids, extending from residue 20 through residue 274 ofSEQ ID NO:53. The nucleotide sequence encoding PCaCD402₅₅, which extendsfrom nucleotide 253 through nucleotide 1017 of SEQ ID NO:52, is denotedherein as nucleic acid molecule nCaCD40₇₆₅, represented by SEQ ID NO:57(the coding strand) and SEQ ID NO:59 (the complement strand).

Sequence analysis was performed with DNAsis™ using the alignmentsettings of: gap penalty set at 5; number of top diagonals set at 5;fixed gap penalty set at 10; k-tuple set at 2; window size set at 5 andfloating gap penalty set at 10. At the amino acid level, PCaCD40₂₇₄shares 65.3%, 50.1%, and 42.3% identity with the CD40 proteins of human,bovine, and mouse, respectively (Stamenkovic et al., EMBO J., vol.8:1403-1410, 1989; Hirano et al., Immunology, vol. 90, pp. 294-300,1997; Grimaldi et al., J. Immunol., vol. 143, pp.3921-3926; Torres andClark, J. Immuno., vol. 148, pp. 620-626). At the nucleotide level,nCaCD40₁₄₂₅ shares 69.3%, 69.4%, and 40.4% identity with the cDNAsequences of human, bovine, and mouse CD40, respectively.

B. Feline CD40 Nucleic Acid Molecules and Proteins

This example describes the isolation and sequencing of certain nucleicaid molecules of the present invention that encode certain feline CD40proteins of the present invention.

A 336-nucleotide feline CD40 nucleic acid molecule, denoted nFeCD40₃₃₆,was amplified from a feline PBMC cDNA library, prepared as described inExample 2, using PCR conditions and primers as described in Example 3A,i.e., a sense primer having SEQ ID NO:128; and an antisense primerhaving SEQ ID NO:129. The resulting PCR product, i.e., nFeCD40₃₃₆, wasshown to have a coding strand the nucleic acid sequence of which isrepresented as SEQ ID NO:60. The reverse complement of SEQ ID NO:60 isreferred to herein as SEQ ID NO:62. Translation of SEQ ID NO:60 suggeststhat nucleic acid molecule nFeCD40₃₃₆ encodes a partial CD40 protein of112 amino acids, denoted herein as PFeCD40₁₁₂, the amino acid sequenceof which is presented in SEQ ID NO:61, assuming an open reading framespanning from nucleotide 1 through nucleotide 336 of SEQ ID NO:60.

Comparison of nucleic acid sequence SEQ ID NO:60 with nucleic acidmolecules reported in GenBank indicates that SEQ ID NO:60 showed themost homology, i.e. 67.2% identity, with a human CD40 gene. Comparisonof amino acid sequence SEQ ID NO:61 with amino acid sequences reportedin GenBank indicates that SEQ ID NO:61 showed the most homology, i.e.about 54.4% identity, with a human CD40 protein. Sequence analysis wasperformed using the GCG GAP program as described above.

Example 4

This example describes the isolation and sequencing of certain canineCD154 (canine CD40 ligand) and feline CD154 (feline CD40 ligand) nucleicacid molecules and proteins of the present invention.

A. Canine CD154 (CD40 ligand) Nucleic Acid Molecules and Proteins

The following describes the isolation and sequencing of certain cDNAnucleic acid molecules encoding certain canine CD154 (CD40 ligand)protein of the present invention.

A canine CD154 nucleic acid molecule of the present invention wasproduced by PCR amplification as follows. A 390-nucleotide canine CD40nucleic acid molecule, denoted nCaCD154₃₉₀, was amplified from a caninePBMC cDNA library, prepared as described in Example 1, using twodegenerate oligonucleotide primers designed in accordance with humanCD154 gene sequences available in GenBank; sense primer, 5′ CCTCAAATTGCGGCACATGT C 3′, denoted herein as SEQ ID NO:130; and antisense primer,5′ CTGTTCAGAG TTTGAGTAAG CC 3′, denoted herein as SEQ ID NO:131. PCRconditions used for canine CD154 cDNA amplification were standardconditions for PCR amplification (Sambrook, et al., ibid.). Theresulting PCR product, i.e., nCaCD154₃₉₀, with a coding strandrepresented by SEQ ID NO:63, was radiolabeled using standard techniquesand used to screen the canine PBMC cDNA library, under the hybridizationconditions described in Example 3. A clone containing a 1878-nucleotidecanine CD154 nucleic acid molecule, denoted nCaCD154₁₈₇₈, was obtained.The nucleic acid sequence of the coding strand of nCaCD154₁₈₇₈ isrepresented as SEQ ID NO:64. The reverse complement of SEQ ID NO:64 isreferred to herein as SEQ ID NO:66. Translation of SEQ ID NO:64 suggeststhat nucleic acid molecule nCaCD154,₈₇₈ encodes a CD154 protein of 260amino acids, denoted herein as PCaCD154₂₆₀, the amino acid sequence ofwhich is presented in SEQ ID NO:65, assuming an open reading framehaving an initiation codon spanning from nucleotide 284 throughnucleotide 286 of SEQ ID NO:64 and a stop codon spanning from nucleotide1064 through nucleotide 1066 of SEQ ID NO:64. The coding region encodingPCaCD154₂₆₀, not including the termination codon, is presented herein asnCaCD154₇₈₀, which has the nucleotide sequence SEQ ID NO:67 (the codingstrand) and SEQ ID NO:68 (the complementary strand).

A putative signal/membrane anchor sequence coding region extends fromnucleotide 284 through nucleotide 430 of SEQ ID NO:64. The proposedsoluble CD154 protein, denoted herein as PCaCD154₂₁₁, represented by SEQID NO:70, contains about 211 amino acids, extending from residue 50though residue 260 of SEQ ID NO:65. The nucleotide sequence encodingPCaCD154₂₁₁, which extends from nucleotide 431 through nucleotide 1063of SEQ ID NO:64, is denoted herein as nucleic acid molecule nCaCD154₆₃₃,represented by SEQ ID NO:69 (the coding strand) and SEQ ID NO:71 (thecomplement strand).

Sequence analysis was performed with DNAsis™ using the alignmentsettings of: gap penalty set at 5; number of top diagonals set at 5;fixed gap penalty set at 10; k-tuple set at 2; window size set at 5 andfloating gap penalty set at 10. At the amino acid level, PCaCD154₂₆₀shares 78.0%, 77.6%, and 67.6% identity with the CD154 proteins ofhuman, bovine, and mouse, respectively (Graf et al., Eur. J. Immunol.,vol. 22, pp. 3191-3194, 1992; Hollenbaugh, et al., EMBO J., vol.11:4313-4321, 1992; Gauchat et al., FEBS lett., vol., 315, pp. 259-266,1993; Mertens et al., Immunogenetics, vol. 42, pp. 430-431; Armitage etal., Nature, vol. 357, pp. 80-82; 1992). At the nucleotide level,nCaCD154₁₈₇₈ shares 81.1%, 81.5%, and 74.4% identity with the sequencesof human, bovine, and mouse CD154 cDNAs, respectively.

B. Feline CD154 (CD40 ligand) Nucleic Acid Molecules and Proteins

This example describes the isolation and sequencing of certain nucleicacid molecules encoding certain feline CD154 (CD40 ligand) proteins ofthe present invention.

A feline CD154 nucleic acid molecule was isolated by PCR amplificationfrom a feline PBMC cDNA library, prepared as described in Example 2,using Amplitaq DNA polymerase (available from PE Applied Biosystems Inc,Foster City, Calif.) under the following PCR protocol: one initialdenaturation step at 95° C. for 5 minutes; then 40 cycles of thefollowing: 94° C. for 45 seconds, then 48° C. for 45 seconds, then 72°C.; for 120 seconds; followed by a final extension at 72° C. for 7minutes. The forward and reverse primers used were based on human CD154cDNA sequences outside the open reading frame in the 5′ and 3′untranslated regions, respectively, so that the open reading frame inthe PCR product contained only feline sequences. The sequence of theforward primer was 5′ GAAGATACCA TTTCAACTTT AACACAGC 3′ SEQ ID NO:132,and that of the reverse primer was 5′ TGCTGTATTG TGAAGACTCC CAGC 3′ SEQID NO:133. PCR products were cloned into the TA cloning vector(available from Invitrogen Corporation, Carlsbad, Calif.), and theresulting clones were sequenced using an ABI Prism™ Model 377 AutomaticDNA Sequencer (available from PE Applied Biosystems Inc.). DNAsequencing reactions were performed using Prism™ dRhodamine TerminatorCycle Sequencing Ready Reaction kits (available from PE AppliedBiosystems Inc.).

The PCR product was sequenced and found to contain 885 nucleotides, andis denoted as nFeCD154₈₈₅. The nucleotide sequence of the coding strandof nFeCD154₈₈₅ is represented herein as SEQ ID NO:72, and its complementis denoted SEQ ID NO:74. Translation of the open reading frame in SEQ IDNO:72 suggests that nFeCD154₈₈₅ encodes a protein containing 260 aminoacids, referred to herein as PFeCD154₂₆₀, the amino acid sequence ofwhich is presented as SEQ ID NO:73, assuming an open reading frame inwhich the first codon spans from nucleotide 29 through nucleotide 31 ofSEQ ID NO:72, and the stop codon spans from nucleotide 809 throughnucleotide 811 of SEQ ID NO:72. The encoded protein has a predictedmolecular weight of 28.6 kDa for the precursor protein and 27.2 kDa forthe mature protein. The coding region encoding PFeCD154₂₆₀, notincluding the termination codon, is presented herein as nFeCD154780,which has the nucleotide sequence SEQ ID NO:75 (the coding strand) andSEQ ID NO:76 (the complementary strand)

A putative signal/membrane anchor sequence coding region extends fromnucleotide 29 through nucleotide 175 of SEQ ID NO:72. The proposedsoluble feline CCD154 protein, denoted herein as PFeCD154₂₁₁,represented by SEQ ID NO:78, contains about 211 amino acids, extendingfrom residue 50 though residue 260 of SEQ ID NO:73. The nucleotidesequence encoding PFeCD154₂₁₁, denoted herein as nFeCD154₆₃₃ whichextends from nucleotide 176 through nucleotide 808 of SEQ ID NO:72, isrepresented herein by SEQ ID NO:77 (the coding strand) and SEQ ID NO:79(the complementary strand).

Comparison of feline CD154 nucleotide and amino acid sequences withthose of other species published in GenBank reveals that the felineCD154 nucleotide sequence SEQ ID NO:75 is 86%, 88% and 75% identical tothe human, bovine and murine CD154 gene sequences, respectively (Genbankaccession number L07414, Z48469 and X56453 respectively). At the aminoacid sequence level, SEQ ID NO:73 is 81%, 82%, and 67% identical to thehuman, bovine and murine CD154 amino acid sequences, respectively.Hydrophobicity analysis of feline CD154 proteins results in a patternsimilar to those of human, bovine and murine CD154 proteins. A putativeN-glycosylation site was identified at position 239 in PFeCD154₂₆₀thatis conserved in the human, bovine and murine amino acid sequences. Fivecysteine residues are present in the feline CD154 protein sequence SEQID NO:73. Four of the five residues, located at positions 72, 84, 177and 217 of PFeCD154₂₆₀, are conserved in all four species and are likelyinvolved in disulfide bond formation. The cysteine residue located atposition 193 of PFeCD154₂₆₀ is present in all but the murine sequence.

Example 5

This example describes the isolation and sequencing of certain canineIL-5 nucleic acid molecules and proteins of the present invention. Thisexample also describes expression of canine IL-5 in a Pichia expressionsystem and the bioactivity of such an expressed protein.

A. Isolation and Sequencing of Canine IL-5 Nucleic Acid Molecules andProteins

A canine IL-5 cDNA nucleic acid molecule encoding a canine IL-5 proteinwas isolated by PCR amplification from a canine PBMC cDNA library(prepared as described in Example 1) using PCR conditions as describedin Example 4B and the following primers. Degenerate oligonucleotideprimers were designed in accordance with conserved regions of human andcat IL-5 gene sequences available in GenBank: sense primer, 5′ATGCACTTTC TTTGCC 3′, denoted herein as SEQ ID NO:134; antisense primer1, 5′ CTGGAGGAAA AKACTTCRAT GATTCTGATA TCTGAAATAT AT 3′, denoted hereinas SEQ ID NO:135; and antisense primer 2, 5′ CTGACYCTTK STTGGSCCTCATTCTCA 3′, denoted herein as SEQ ID NO:136, where K was G or T, R waseither A or G, S was either G or C, and Y was either T or C.

An about 610-nucleotide canine IL-5 nucleic acid molecule, denotednCaIL-5₆₁₀, was obtained using primers having SEQ ID NO:134 and SEQ IDNO:135, respectively. The sequence of the coding strand of nCaIL-5₆₁₀ isrepresented herein as SEQ ID NO:80. The reverse complement of SEQ IDNO:80 is referred to herein as SEQ ID NO:82. Translation of SEQ ID NO:80suggests that nucleic acid molecule nCaIL-5₆₁₀ encodes an IL-5 proteinof 134 amino acids, denoted herein as PCaIL-5₁₃₄, the amino acidsequence of which is presented in SEQ ID NO:81, assuming an open readingframe having an initiation codon spanning from nucleotide 29 throughnucleotide 31 of SEQ ID NO:80 and a stop codon spanning from nucleotide431 through nucleotide 433 of SEQ ID NO:80. The coding region encodingPCaIL-13₁₃₄, not including the termination codon, is presented herein asnCaIL-5₄₀₂, which has the nucleotide sequence SEQ ID NO:83 (the codingstrand) and SEQ ID NO:84 (the complementary strand).

At about 488-nucleotide fragment, denoted herein as nCaIL-5₄₈₈, isolatedby PCR with primers having SEQ ID NO:134 and SEQ ID NO:136,respectively, corresponds to nucleotide 1 through nucleotide 488 ofnCaIL-5₆₁₀.

A putative signal sequence coding region extends from nucleotide 29through nucleotide 85 of SEQ ID NO:80. The proposed mature protein,denoted herein as PCaIL-5₁₁₅, represented by SEQ ID NO:86, containsabout 115 amino acids, extending from residue 20 though residue 134 ofSEQ ID NO:81. The nucleotide sequence encoding PCaIL-5₁₁₅, which extendsfrom nucleotide 86 through nucleotide 430 of SEQ ID NO:80, is denotedherein as nucleic acid molecule nCaIL-5₃₄₅, represented by SEQ ID NO:85(coding strand) and SEQ ID NO:87 (the complement strand).

Sequence analysis was performed with DNAsis™ using the alignmentsettings of: gap penalty set at 5; number of top diagonals set at 5;fixed gap penalty set at 10; k-tuple set at 2; window size set at 5 andfloating gap penalty set at 10. At the amino acid level, PCaIL-5₁₃₄shared 82.8% and 57.4% identity with feline and human IL-5 proteins,respectively (Padrid et al., Am. J. Vet. Res., vol. 59, pp. 1263-1269,1998; Azuma et al., Nucleic Acids Res., vol. 14, pp. 9149-9158, 1986).At the nucleotide level, nCaIL-5₆₁₀ shared 81.7% and 75% identity withthe cDNA sequences of the feline and human IL-5, respectively.

B. Expression of Canine IL-5 in Pichia

This example describes the expression in Pichia of a canine IL-5 cDNAfragment, namely a canine IL-5 nucleic acid molecule denoted nCaIL-5₃₄₈,the coding strand of which consists of nucleotides 86-433 of SEQ IDNO:80, and as such, encodes a predicted mature canine IL-5 proteinhaving SEQ ID NO:86. Nucleic acid molecule nCaIL-5₃₄₈, was PCR amplifiedfrom nCaIL-5₆₁₀ using sense primer 5′ GGGCTCGAGA AAAGATTTGC TGTAGAAAATCCCATG 3′ deonted herein as SEQ ID NO:137, with nucleotides 16-36corresponding to nucleotides 86-106 of SEQ ID NO:80; and antisenseprimer 5′ CCCGCGGCCG CTCAACTTTC CGGTGTCCAC TC 3′, denoted herein as SEQID NO:138, with nucleotides 12-32 corresponding to the reversecomplement of nucleotides 413-433 of SEQ ID NO:80. To facilitatecloning, an XhoI site (shown in bold) was added to the sense primer anda NotI site (shown in bold) was added to the antisense primer. ThePCR-amplified fragment was digested with restriction endonucleases XhoIand NotI, gel purified and ligated into pPICZαA plasmid vector,available from Invitrogen, that has been digested by Xho I and Not I andgel purified, to produce recombinant molecule pPICZαA-nCaIL-5₃₄₈. Theinsert in the recombinant molecule was verified by DNA sequencing Therecombinant molecule was used to transform Pichia pastoris strain X-33by electroporation to produce recombinant cellPichia-pPICZαA-nCaIL-5₃₄₈. Recombinant cell Pichia-pPICZαA-nCaIL-5₃₄₈was cultured using techniques known to those skilled in the art and IL-5expression was induced with methanol. The supernatant was recovered andsubmitted to SDS-PAGE. Silver staining of the resultant gel indicated aband of about 18 kDa only seen in the supernatant of Pichia transformedwith recombinant molecule pPICZαA-nCaIL-5₃₄₈.

C. Bioactivity of Pichia-expressed Canine IL-5

The following describes a bioassay to detect the expression of canineIL-5 by stimulating the proliferation of TF-1 cells.

TF-1 cells, grown and maintained as described in Example 1E, wereextensively washed to remove rhuGM-CSF, and then added at approximately1×10⁴ cells per well to 96-well flat bottom plates. Pichia-expressedcanine IL-5, produced as described in Example 5B, was dialyzed overnightat 4° C. against Phosphate Buffered Saline, diluted to the appropriateconcentration in TCM-TF-1 without rhuGM-CSF and filter sterilized. Cellsand Pichia-produced canine IL-5 were incubated for 48 hours in 5% CO₂ at37° C., then pulsed, incubated, harvested and counted as described inExample 1E. The results are shown in Table 5.

TABLE 5 Stimulation of proliferation of TF-1 with Pichia-expressedcanine IL-5 1/dilution Counts per minute 2 44,885 4 101,564 8 81,161 1659,384 32 40,508 64 15,948 128 6,634 256 2,441 Media (no IL-5) 172

Table 5 shows that canine IL-5 expressed by Pichia is biologicallyactive, as demonstrated by its ability to stimulate proliferation ofTF-1 cells.

Example 6

This example describes the isolation and sequencing of certain canineIL-13 nucleic acid molecules and proteins of the present invention. Thisexample also describes expression of canine IL-13 in E. coli andbioactivity of such an expressed protein.

A. Isolation and Sequencing of Canine IL-13 Nucleic Acid Molecules andProteins

A canine IL-13 cDNA nucleic acid molecule encoding a canine IL-13protein was isolated by PCR amplification from a canine PBMC cDNAlibrary (prepared as described in Example 1) using the following primersand PCR conditions: Degenerate oligonucleotide primers were designed inaccordance with conserved regions of human and cat IL-5 gene sequencesavailable in GenBank: sense primer, 5′ GTCMTKGCTC TYRCTTGCCT TGG 3′,denoted herein as SEQ ID NO:139; antisense primer 1, 5′ AAASTGGGCYACYTCGATTT TGG 3′, denoted herein as SEQ ID NO:140; antisense primer 2,5′ GTGATGTTGM YCAGCTCCTC 3′, denoted herein as SEQ ID NO:141, where Mwas either A or C, K was G or T, R was either A or G, S was either G orC, and Y was either T or C. PCR conditions used were as follows: Oneinitial denaturation step at 95° C. for 3 minutes; then 38 cycles of thefollowing: 94° C. for 30 seconds, 51.8° C. for 45 seconds, then 72° C.for 105 seconds; then a final extension at 72° C. for 5 minutes.

An about 272-nucleotide canine IL-13 nucleic acid molecule, denotednCaIL-13₂₇₂ and having a coding strand represented by SEQ ID NO:89, wasPCR amplified using primers having nucleic acid sequences of SEQ IDNO:139 and SEQ ID NO:140, respectively. An about 166-nucleotide canineIL-13 nucleic acid molecule, denoted nCaIL-13₁₆₆ and having a codingstrand represented by SEQ ID NO:88, was isolated using primers nucleicacid sequences of SEQ ID NO:142 (see Example 2B) and SEQ ID NO:141,respectively. Nucleic acid molecules nCaIL-13₂₇₂ and nCaIL-13₂₇₂ form aoverlapping composite fragment of 383 nucleotides, denoted nCaIL-13₃₈₃.Two canine IL-13 specific primers (i.e., sense primer, 5′ ATGGCGCTCTGGTTGACTGT 3′, denoted herein as SEQ ID NO:143; and antisense primer, 5′GGCTTTTGAG AGCACAGTGC 3′, denoted herein as SEQ ID NO:144) were derivedfrom nCaIL-13₃₈₃ and were used to isolate a 278-nucleotide fragment,denoted nCaIL-13₂₇₈ and having a coding strand represented by SEQ IDNO:90. Nucleic acid molecule nCaIL-13₂₇₈ was radiolabeled and used toscreen the canine PBMC cDNA library under the following hybridizationconditions: hybridization took place in 6×SSC, 5×Denhardt's solution,0.5% SDS, 100 μg/ml single stranded DNA, 100 μg/ml tRNA, for 36 hours at60° C.; the final wash solution was 0.1% SDS, 0.125×SSC at 60° C. for 30minutes. Two clones were selected, namely clone 80 and clone 78.

Sequence analysis of Clone 80 indicated that the clone includes an about1302-nucleotide canine IL-13 nucleic acid molecule referred to herein asnCaIL-13₁₃₀₂, the coding strand of which was shown to have nucleic acidsequence SEQ ID NO:91. The reverse complement of SEQ ID NO:91 isreferred to herein as SEQ ID NO:93. Translation of SEQ ID NO:91 suggeststhat nucleic acid molecule n nCaIL-13₁₃₀₂encodes an IL-13 protein of 131amino acids, denoted herein as PCaIL-13₁₃₁, the amino acid sequence ofwhich is presented in SEQ ID NO:92, assuming an open reading framehaving an initiation codon spanning from nucleotide 52 throughnucleotide 54 of SEQ ID NO:91 and a stop codon spanning from nucleotide445 through nucleotide 447 of SEQ ID NO:91. The coding region encodingPCaIL-13₁₃₁, not including the termination codon, is presented herein asnCaIL-13₃₉₃, which has the nucleotide sequence SEQ ID NO:94 (the codingstrand) and SEQ ID NO:95 (the complementary strand).

A putative signal sequence coding region extends from nucleotide 52 tonucleotide 111 of SEQ ID NO:91. The proposed mature protein, denotedherein as PCaIL-13₁₁₁, represented by SEQ ID NO:97, contains 111 aminoacids, extending from residue 21 through residue 131 of SEQ ID NO:91.The nucleotide sequence encoding PCaIL-13₁₁₁, which extends fromnucleotide 112 through nucleotide 444 of SEQ ID NO:91, is denoted hereinas nucleic acid molecule nCaIL-13₃₃₃, represented by SEQ ID NO:96(coding strand) and SEQ ID NO:98 (the complement strand).

Sequence analysis of Clone 78 indicated that the clone includes an about1269-nucleotide canine IL-13 nucleic acid molecule referred to herein asnCaIL-13₁₂₆₉, the coding strand of which was shown to have nucleic acidsequence SEQ ID NO:99. The reverse complement of SEQ ID NO:99 isreferred to herein as SEQ ID NO:101. Translation of SEQ ID NO:99suggests that nucleic acid molecule nCaIL-13₁₂₆₉ encodes an IL-13protein of 130 amino acids, denoted herein as PCaIL-13₁₃₀, the aminoacid sequence of which is presented in SEQ ID NO:100, assuming an openreading frame having an initiation codon spanning from nucleotide 57through nucleotide 59 of SEQ ID NO:99 and a stop codon spanning fromnucleotide 447 through nucleotide 449 of SEQ ID NO:99. The coding regionencoding PCaIL-13₁₃₀, not including the termination codon, isrepresented herein as nCaIL-13₃₉₀, which has the nucleotide sequence SEQID NO:102 (the coding strand) and SEQ ID NO:103 (the complementarystrand). PCaIL-13₁₃₀ is missing one amino acid compared to PCaIL-13₁₃₃,namely amino acid position Q97 of PCaIL-13₁₃₃.

A putative signal sequence coding region extends from nucleotide 57 tonucleotide 116 of SEQ ID NO:99. The proposed mature protein, denotedherein as PCaIL-13₁₁₀, represented by SEQ ID NO:105, contains 110 aminoacids, extending from residue 21 though residue 130 of SEQ ID NO:100.The nucleotide sequence encoding PCaIL-13₁₁₀, which extends fromnucleotide 117 through nucleotide 446 of SEQ ID NO:99, is denoted hereinas nucleic acid molecule nCaIL-13₃₃₀, represented by SEQ ID NO:104(coding strand) and SEQ ID NO:106 (the complement strand).

Sequence analysis was performed with DNAsis™ using the alignmentsettings of: gap penalty set at 5; number of top diagonals set at 5;fixed gap penalty set at 10; k-tuple set at 2; window size set at 5 andfloating gap penalty set at 10. At the amino acid level, PCaIL-13₁₃₁shared 61.7%, 39.6%, 36.6% identity with the IL-13 proteins of human,mouse, and rat (Brown et al., J. Immunol., vol. 142, pp. 679-687, 1989;Lakkis et al., Biochem. Biophys. Res. Commun., Vol. 197, pp. 612-618,1993; McKenzie et al., Proc. Natl Acad. Sci. USA, vol. 90, pp.3735-3739, 1993; Minty et al., Nature, vol. 362, pp. 248-250, 1993),respectively. At the nucleotide level, nCaIL-13₁₃₀₂ shared 56.0%, 57.1%,and 45.9% identity with the sequences of human, rat, and mouse IL-13cDNAs, respectively.

B. Expression of Canine IL-13 in E. coli

This examples describes the expression in E. coli of a canine IL-13 cDNAfragment, namely a canine IL-13 nucleic acid molecule denotednCaIL-13₃₃₆, the coding strand of which consists of nucleotides 112-447of SEQ ID NO:91, and as such, encodes a predicted mature canine IL-13protein having SEQ ID NO:97. Nucleic acid molecule nCaIL-13₃₃₆ was PCRamplified from nCaIL-13₁₃₀₂ using sense primer 5′ CCCCATATGA GCCCTGTGACTCCCTCCCC 3′ denoted herein as SEQ ID NO:145, with nucleotides 10-29corresponding to nucleotides 112-1131 of SEQ ID NO:91; and antisenseprimer 5′ GGGGAATTCT CATCTGAAAT TTCCATGGCG 3′, denoted herein as SEQ IDNO:146, with nucleotides 10-30 corresponding to the reverse complementof nucleotides 427-447 of SEQ ID NO:91. To facilitate cloning, an NdeIsite (shown in bold) was added to the sense primer and an EcoRI site(shown in bold) was added to the antisense primer. The resulting PCRfragment was digested with restriction endonucleases NdeI and EcoRI, gelpurified and ligated into λcro plasmid vector, the production of whichis described in U.S. Pat. No. 5,569,603 by Tripp et al., issued Oct. 29,1996, that had been digested by NdeI and EcoRI and gel purified toproduce recombinant molecule pλcro-nCaIL-13₃₃₆. The insert in therecombinant molecule was verified by DNA sequencing. Recombinantmolecule pλcro-nCaIL-13₃₃₆ was used to transform E. coli strain HCE101(BL21), thereby producing BL21-pλcro-nCaIL-13₃₃₆. PCaIL-13₁₁₁ wasproduced under conditions as described in U.S. Pat. No. 5,569,603,ibid., protein expression being induced by switching the fermentationtemperature from 32° C. to 42° C. SDS-PAGE and Commassie blue staininganalysis indicated that a band of about 11 kD was only produced byinduced BL21-pλcro-nCaIL-13₃₃₆ recombinant cells. The 11-kD band showeda positive reaction with a rabbit polyclonal antibody against humanIL-13 (available from PeproTech Inc, Rocky Hill, N.J.), indicatingexpression of canine IL-13 in E. coli.

C. Bioactivity of E. coli-expressed Canine IL-13

The following describes a bioassay to detect the expression of canineIL-13 by stimulating the proliferation of TF-1 cells.

TF-1 cells, grown and maintained as described in Example 1E, wereextensively washed to remove rhuGM-CSF, and then added at approximately1×10⁴ cells per well to 96-well flat bottom plates. E. coli-producedPCaIL-13₁₁₁, produced as described in Example 6B, was dialyzed overnightat 4° C. against Phosphate Buffered Saline, diluted to the appropriateconcentration in TCM-TF-1 without rhuGM-CSF and filter sterilized. Cellsand E. coli-produced PCaIL-13₁₁₁ were incubated for 48 hours in 5% CO₂at 37° C., then pulsed, incubated, harvested and counted as described inExample 1E. The results are shown in Table 6.

TABLE 6 Stimulation of proliferation of TF-1 cells with E. coliPCaIL-13₁₁₁ Concentration E. coli PCaIL-13₁₁₁ (ng/ml) Counts per minute1000 126,203 500 77,893 250 57,781 125 40,491 62.5 26,115 31.3 7,04215.6 8,713 0 991

Table 6 shows that canine IL-13 expressed by E. coli is biologicallyactive, as demonstrated by its ability to stimulate proliferation ofTF-1 cells.

Example 7

This example describes the isolation and sequencing of feline interferonalpha nucleic acid molecules and proteins of the present invention. Thisexample also describes expression of feline interferon alpha proteins ofthe present invention in E. coli and mammalian cells as well as thebioativites of the resulting proteins.

A. Isolation and Sequencing of Feline IFN-alpha Nucleic Acids andProteins

Feline IFN-alpha nucleic acid molecules were PCR amplified from a felinecDNA library as follows. Total RNA was isolated from cat (kitten)mesenteric lymph node cells stimulated with PMA (phorbol myristateacetate) for 48 hours using Tri Reagent™ (available from MolecularResearch Center, Cincinnati, Ohio). cDNA was made from the RNA using thecDNA synthesis kit containing Ready to Go -You Prime First-Strand Beads™(available from Amersham Pharmacia Biotech, Piscataway, N.J.). Analiquot of this cDNA was used as a template to isolate a felineIFN-alpha nucleic acid molecule by PCR amplification using Amplitaq DNApolymerase™ (available from PE Applied Biosystems Inc, Foster City,Calif.) and the following primers and conditions. The sequence of theforward primer was 5′ ATGGCGCTGC CCTCTTCCTT CTTG 3′ (SEQ ID NO:143), andthat of the reverse primer was 5′ TCATTTCTCG CTCCTTAATC TTTTCTGC 3′ (SEQID NO:148). The following PCR protocol was used: one initialdenaturation step at 95° C. for 5 minutes; then 43 cycles of thefollowing: 94° C. for 45 seconds, then 47° C. for 45 seconds, then 72°C. for 120 seconds; followed by a final extension at 72° C. for 7minutes. PCR products were cloned into the TA cloning vector (availablefrom Invitrogen Corporation) and the clones were sequenced using an ABIPrism™ Model 377 Automatic DNA Sequencer (available from PE AppliedBiosystems Inc.). DNA sequencing reactions were performed using Prism™dRhodamine Terminator Cycle Sequencing Ready Raction kits (availablefrom PE Applied Biosystems Inc.). Five PCR products were generated andsequenced. These products were included, respectively, in Clones #1, #2,#3, #5, and #6.

Clone #2 includes a feline IFN-alpha nucleic acid molecule that isrepresented herein as nFeIFNα_(567a), the coding strand of which wasshown to have a nucleic acid sequence denoted herein as SEQ ID NO:107.The complement of SEQ ID NO:107 is represented herein by SEQ ID NO:109.Translation of SEQ ID NO:107 suggests that nFeIFNα_(567a) encodes aprotein containing 189 amino acids, referred to herein asPFeIFNα_(189a), with an amino acid sequence denoted SEQ ID NO:108. Theopen reading frame of SEQ ID NO:107 is assumed to be the following: thefirst codon spans from nucleotide 1 through nucleotide 3 and the lastcodon before the stop codon spans from nucleotide 565 to nucleotide 567of SEQ ID NO:107. The encoded protein has a predicted molecular weightof 21 kDa. The putative signal peptide cleavage site occurs betweenamino acid positions 23 and 24, based on homology with the human andcanine interferon-alpha proteins. The proposed mature protein (i.e.feline IFNα protein from which the signal sequence has been cleaved),denoted herein as PFeIFNα_(166a), contains about 166 amino acids,extending from residue 24 to residue 166 of SEQ ID NO:108; the aminoacid sequence is denoted herein as SEQ ID NO:114. The nucleic acidmolecule encoding PFeIFNα_(166a) is denoted herein as nFeIFNα_(498a),the coding strand of which is represented by SEQ ID NO:113, and thecomplementary strand of which is represented by SEQ ID NO:115. Aputative N-glycosylation site and an interferon alpha-beta-delta familysignature motif are present at amino acid positions 102 and 145,respectively, of PfeIFNα_(189a).

Clone #3 includes a feline IFN-alpha nucleic acid molecule that isrepresented herein as nFeIFNα_(567b), the coding strand of which wasshown to have a nucleic acid sequence denoted herein as SEQ ID NO:110.The complement of SEQ ID NO:110 is represented herein by SEQ ID NO:112.Translation of SEQ ID NO:110 suggests that nFeIFNα_(567b) encodes aprotein containing 189 amino acids, referred to herein asPFeIFNα_(189b), with an amino acid sequence denoted SEQ ID NO:111. Theopen reading frame of SEQ ID NO:110 is assumed to be the following: thefirst codon spans from nucleotide 1 through nucleotide 3 and the lastcodon before the stop codon spans from nucleotide 565 through nucleotide567 of SEQ ID NO:110. The encoded protein has a predicted molecularweight of 21 kDa. The putative signal peptide cleavage site occursbetween amino acid positions 23 and 24, based on homology with the humanand canine interferon-alpha proteins. The proposed mature protein (i.e.feline IFNα protein from which the signal sequence has been cleaved),denoted herein as PFeIFNα_(166b), contains about 166 amino acids,extending from residue 24 to residue 166 of SEQ ID NO:111; the aminoacid sequence is denoted herein as SEQ ID NO:117. The nucleic acidmolecule encoding PFeIFNα_(166b) is denoted herein as nFeIFNα_(498b),the coding strand of which is represented by SEQ ID NO:116, andcomplementary strand of which is represented by SEQ ID NO:118. Aputative N-glycosylation site and an interferon alpha-beta-delta familysignature motif are present at amino acid positions 102 and 145,respectively, of PFeIFNα_(189b).

Clone #1 includes a feline IFN-alpha nucleic acid molecule that isrepresented herein at nFeIFNα_(567c), the coding strand of which wasshown to have a nucleic acid sequence denoted herein as SEQ ID NO:155.The complement of SEQ ID NO:155 is represented herein by SEQ ID NO:157.Translation of SEQ ID NO:155 suggests that nFeIFNα_(567c) encodes aprotein containing 189 amino acids, referred to herein asPFeIFNα_(890c), with an amino acid sequence denoted SEQ ID NO:156. Theopen reading frame of SEQ ID NO:155 is assumed to be the following: thefirst codon spans from nucleotide 1 through nucleotide 3 and the lastcodon before the stop codon spans from nucleotide 565 to nucleotide 567of SEQ ID NO:155. The encoded protein has a predicted molecular weightof 21 kDa. The putative signal peptide cleavage site occurs betweenamino acid positions 23 and 24, based on homology with the human andcanine interferon-alpha proteins. The proposed mature protein (i.e.feline IFNa protein from which the signal sequence has been cleaved),denoted herein as PFeIFNa_(166c), contains about 166 amino acids,extending from residue 24 to residue 166 of SEQ ID NO:156; the aminoacid sequence is denoted herein as SEQ ID NO:159. The nucleic acidmolecule encoding PFeIFNa_(166c) is denoted herein as nFeIFNa_(498c),the coding strand of which is represented by SEQ ID NO:158, and thecomplementary strand of which is represented by SEQ ID NO:160. Aputative N-glycosylation site and an interferon alpha-beta-delta familysignature motif are present at amino acid positions 102 and 145,respectively, of PfeIFNa_(189c).

Clone #5 includes a feline IFN-alpha nucleic acid molecule that isrepresented herein as nFeIFNa_(582d), the coding strand of which wasshown to have a nucleic acid sequence denoted herein as SEQ ID NO:161.The complement of SEQ ID NO:161 is represented herein by SEQ ID NO:163.Translation of SEQ ID NO:161 suggests that nFeIFNa_(582d) encodes aprotein containing 194 amino acids, referred to herein asPFeIFNa_(194d), with an amino acid sequence denoted SEQ ID NO:162. Theopen reading frame of SEQ ID NO:161 is assumed to be the following: thefirst codon spans from nucleotide 1 through nucleotide 3 and the lastcodon before the stop codon spans from nucleotide 580 through nucleotide582 of SEQ ID NO:161. The encoded protein has a predicted molecularweight of 21.5 kDa. The putative signal peptide cleavage site occursbetween amino acid positions 23 and 24, based on homology with the humanand canine interferon-alpha proteins. The proposed mature protein (i.e.feline IFNa protein from which the signal sequence has been cleaved),denoted herein as PFeIFNa_(171d), contains about 171 amino acids,extending from residue 24 to residue 171 of SEQ ID NO:162; the aminoacid sequence is denoted herein as SEQ ID NO:165. The nucleic acidmolecule encoding PFeIFNa_(171d) is denoted herein as nFeIFNa_(513d),the coding strand of which is represented by SEQ ID NO:164, and thecomplementary strand of which is represented by SEQ ID NO:166. Aputative N-glycosylation site and an interferon alpha-beta-delta familysignature motif are present at amino acid positions 102 and 145,respectively, of PFeIFNa_(194d).

Clone #6 includes a feline IFN-alpha nucleic acid molecule that isrepresented herein as nFeIFNa_(567e), the coding strand of which wasshown to have a nucleic acid sequence denoted herein as SEQ ID NO:167.The complement of SEQ ID NO:167 is represented herein by SEQ ID NO:169.Translation of SEQ ID NO:167 suggests that nFeIFNa_(567e) encodes aprotein containing 189 amino acids, referred to herein asPFeIFNa_(189e), with an amino acid sequence denoted SEQ ID NO:168. Theopen reading frame of SEQ ID NO:167 is assumed to be the following: thefirst codon spans from nucleotide 1 through nucleotide 3 and the lastcodon before the stop codon spans from nucleotide 565 to nucleotide 567of SEQ ID NO:167. The encoded protein has a predicted molecular weightof 21 kDa. The putative signal peptide cleavage site occurs betweenamino acid positions 23 and 24, based on homology with the human andcanine interferon-alpha proteins. The proposed mature protein (i.e.feline IFNa protein from which the signal sequence has been cleaved),denoted herein as PFeIFNa_(166e), contains about 166 amino acids,extending from residue 24 to residue 166 of SEQ ID NO:167; the aminoacid sequence is denoted herein as SEQ ID NO:171. The nucleic acidmolecule encoding PFeIFNa_(166e), is denoted herein as nFeIFNa_(498e),the coding strand of which is represented by SEQ ID NO:170, and thecomplementary strand of which is represented by SEQ ID NO:172. Aputative N-glycosylation site and an interferon alpha-beta-delta familysignature motif are present at amino acid positions 102 and 145,respectively, of PfeIFNa_(189e).

Comparison of the nucleic acid sequences of the five feline IFN-alphanucleic acid molecules of the present invention indicated that thesequences, while being very similar (i.e., encoded proteins sharing fromabout 96% to 99% identity), exhibited several differences. Thedifferences in nucleic acid sequences and deduced amino acid sequencesare summarized in Table 7. The left hand column indicates the change atthe nucleotide or amino acid level, as appropriate, and the “X”s in theother columns indicate which clones include such changes. For example,feline IFN-alpha protein PfeIFNa_(194d) (having SEQ ID NO:161) has fiveextra amino acids (namely IHPED) inserted at position at 139 as comparedto feline IFN-alpha proteins PfeIFNa_(189a) (SEQ ID NO:108),PfeIFNa_(189b) (SEQ ID NO:111), PfeIFNa_(189c) (SEQ ID NO:155) orPfeIFNa_(189e) (SEQ ID NO:167). Other variations, i.e., nucleotidesubstitutions, some of which lead to amino acid variations, are alsoindicated in Table 7.

TABLE 7 Comparison of feline IFN-alpha nucleic acid molecules andproteins Amino acid Clone Clone Clone Clone Clone Changes #1 #2 #3 #5 #65 amino acid deletion X X X X S₁₈ to S₁₈ X (TCC to TCT) C₅₂ to C₅₂ X(TGT to TGC) R₅₆ to R₅₆ X (AGA to AGG) N₅₇ to S₅₇ X X (AAT to AGT) F₆₆to F₆₆ X X (TTC to TTT) A₇₄ to A₇₄ X (GCC to GCT) K₈₆ to E₈₆ X (AAG toGAG) R₁₁₅ to W₁₁₅ X X (CGG to TGG) L₁₂₅ to V₁₂₅ X X X (CTG to GTG) L₁₂₅to M₁₂₅ X X (CTG to ATG) L₁₃₅ to L₁₃₅ X X X X (CTG to CTC) L₁₄₁ to L₁₄₁X (ATC to CTC)

Feline IFN-alpha proteins of the present invention PFeIFNα_(189a),PfeIFNα_(189b), PFeIFNα_(189c), and PFeIFNα_(189e) are five amino acidsshorter than the GenBank entry for feline IFN-omega, accession #E02521,while IFN-alpha protein PFeIFNα_(194d) of the present invention has thesame number of amino acids as the feline IFN-omega reported in GenBank.In addition, there are: 3 non-conservative and 2 conservative changes atthe amino acid level between this GenBank entry and SEQ ID NO:108(PFeIFNα_(189a)); 4 non-conservative and 3 conservative changes at theamino acid level between this GenBank entry and SEQ ID NO:111(PfeIFNα_(189b)); 4 non-conservative and 3 conservative changes at theamino acid level between this GenBank entry and SEQ ID NO:156(PFeIFNa_(189c)); 2 non-conservative and 2 conservative changes at theamino acid level between this GenBank entry and SEQ ID NO:162(PfeIFNa_(194d); and 1 non-conservative and 5 conservative changes atthe amino acid level between this GenBank entry and SEQ ID NO:168(PFeIFNa_(189e)).

The lengths of SEQ ID NO:108 and SEQ ID NO:111, when compared with thoseof IFN-alpha proteins of other species, are two amino acids longer thanpublished canine interferon-alpha subtype 1, 2 and 3 sequences, twoamino acids longer than published human interferon-alpha type 1,B,D, F,and J sequences, three amino acids longer than the published humaninterferon-alpha sequence type A sequence and two amino acids longerthan published murine interferon-alpha type B, 8, 7, 11, and 19sequences. The lengths of SEQ ID NO:156 and SEQ ID NO:168, when comparedwith those of IFN-alpha proteins of other species, are two amino acidslonger than published canine interferon-alpha subtype 1, 2 and 3sequences, two amino acids longer than published human interferon-alphatype 1,B,D, F, and J sequences, three amino acids longer than thepublished human interferon-alpha sequence type A sequence and two aminoacids longer than published murine interferon-alpha type B, 8, 7, 11,and 19 sequences. The length of SEQ ID NO:162, when compared with thoseof IFN-alpha proteins of other species, are seven amino acids longerthan published canine interferon-alpha subtype 1, 2 and 3 sequences,seven amino acids longer than published human interferon-alpha type1,B,D, F, and J sequences, eight amino acids longer than the publishedhuman interferon-alpha sequence type A sequence and seven amino acidslonger than published murine interferon-alpha type B, 8, 7, 11, and 19sequences.

B. Expression of Feline IFN-alpha Proteins in Mammalian Cells

This example describes the expression of the feline IFN-alpha proteinsof the present invention in Chinese hamster ovary (CHO) cells.

Feline IFN-alpha nucleic acid molecule PCR products were amplified fromnFeIFNα_(567a), nFeIFNα_(567b), nFeIFNα_(567c), nFeIFNα_(582d), andnFeIFNα_(567e) using Pfu DNA polymerase™ (available from Stratagene, LaJolla, Calif.) and the following primes and conditions. The sequence ofthe forward primer was 5′ ATTAGGATCC ATGGCGCTGC CCTCTTCCT 3′ (SEQ IDNO:173), and that of the reverse primer was 5′ GCCTCTAGAC TGTCATTTCTCGCTCCTTAA TCTTTTCTGC 3′ (SEQ ID NO:174). The following PCR protocol wasused: one initial denaturation step at 95° C. for 5 minutes; then 30cycles of the following: 94° C. for 30 seconds, then 50° C. for 30seconds, then 72° C. for 90 seconds; followed by a final extension at72° C. for 7 minutes.

Each of the five PCR products was ligated into a CMV-Int A-kan+(amp)expression vector using techniques similar to those described in Example1Bii to produce recombinant molecules in which feline IFN-alpha nucleicacid molecules were operatively linked to transcription controlsequences. It is to be noted that CMV-Int A-kan⁺(amp) vector is similarto the pCMV-Int A plasmid vector described in Example 1Bii except thatthe ampicillin resistance gene open reading frame has been disrupted bythe insertion of the kanamycin resistance gene. The feline IFN-alphanucleic acid molecules in the recombinant molecules were sequenced usingan ABI Prism™ Model 377 Automatic DNA Sequencer (available from PEApplied Biosystems Inc.). DNA sequencing reactions were performed usingPrism™ dRhodamine Terminator Cycle Sequencing Ready Reaction kits(available from PE Applied Biosystems Inc.). The sequence data indicatedthat there was no changes introduced during the PCR amplification orligation in any of the nucleic acid molecules.

Using techniques similar to those described elsewhere herein. CHO cellswere transiently transfected with each of the five recombinant moleculesencoding a subtype of feline IFN-alpha protein using Lipofectamine™(available from Life technologies, Inc.) resuting in recombinant cellsexpressing feline IFN-alpha subtype proteins of the present invention.The cells and culture supernatants were harvested 48 hours later andWestern analysis was done using both pellets and the supernatants fromeach transfection. The detecting antibody was an anti-human IFN-alpha-Aantibody (available from Accurate Chemical and Scientific Corporation,Westbury, N.Y.). The Western analysis indicated that each of the fivefeline IFN-alpha nucleic acid molecule-containing recombinant cellsexpressed a corresponding feline IFN-alpha subtype protein which wassecreted into the tissue culture supernatant and recognized by theantibody against human IFN-alpha-A. The migration patterns of each ofthe CHO cell-expressed feline IFN-alpha subtype proteins suggested thateach of the proteins is glycosylated.

C. Bioactivity of Mammalian-expressed Feline-IFN Alpha Proteins

(i) The antiviral activity of the five CHO-expressed feline IFN-alphasubtype proteins, produced as described in Example 7B, was tested usingthe following protocol: Crandell feline kidney (CRFK) cells were treatedfor 24 hours, using procedures known to those skilled in the art, withor without IFN-alpha tissue culture supernatants, produced as describedin Example 7B. The cells were then infected with feline calicivirus andcytophathic effects induced by the virus were assessed 12 to 14 hourslater using techniques known to those skilled in the art. The celllayers were fixed in methanol, stained with crystal violet and examinedunder the microscope or processed for the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.The MTT assay was conducted as follows. After viral infection, theinfected cells were washed with PBS. A volume of MTT stock solution (5mg/ml in PBS) equal to one-tenth of the original culture volume wasadded to each well being assayed and incubated at 37° C. for 3 to 4 hr.The MTT solution was removed, and acidified isopropanol ( ). 1 N HCl inabsolute isopropanol) was added to the wells to solubilize the converteddye. The absorbance of the converted dye was measured at 570 nm using aplate reader. Each of the five IFN-alpha subtype proteins demonstratedanti-viral activity. Pre-treatment with any of the subtypes of IFN-alphaproteins of the present invention resulted in significant reduction inthe virus-induced cytopathic effect.

(ii) The CHO cell-expressed feline IFN-alpha subtype proteins were alsotested for their ability to inhibit granulocyte-macrophage colonystimulating factor-induced proliferation of TF-1 cells using an assaysimilar to that described in Example 1E, but with the followingmodification:. For the assay, the cells were washed and TCM-TF-1 mediumcontaining a suboptimal amount of GM-CSF (i.e., 0.4 ng/ml) was addedalong with the appropriate dilutions of the designated IFN-alphaproteins. The results are shown in Table 8 for feline IFN-alpha proteinsexpressed as described in Example 7B, lanes labeled Clone #1, Clone #2,Clone #3, Clone #5 and Clone #6, respectively; supernatant from aculture of CHO cells transfected with only the vector described inExample 7B, lane labeled vector; E. coli-expressed feline IFN-alphaprotein PFeIFNa_(166c) produced as described in Example 7D, lane labeledE. coli-expressed; and recombinant human IFN-alpha, lane labeled humanIFN-alpha. Media alone gave a reading of 128 and recombinant GM-CSFalone gave a reading of 96080.

TABLE 8 Inhibition of TF-1 cell production by CHO cell-expressed felineIFN-alpha proteins Clone Clone Clone Clone Clone E. coli Human Dilution#1 #2 #3 #5 #6 Vector expressed IFN alpha 2 15077 7914 21173 15218 1325653585 19541 559 4 18318 23515 41488 43449 31618 64722 56315 10412 822484 25823 48487 40438 43896 83092 80646 21710 16 42138 34274 7214566266 48775 102423 97255 23585 32 81248 52847 63264 95256 64 74613 4384858533 88172 70596 141821 129556 45907 128 59360 48901 48701 54623 90092155960 151946 40402 256 75788 54017 37391 59849 83022 119491 12379439299

Table 8 demonstrates that CHO cell-expressed and E. coli-expressedfeline IFN-alpha subtype proteins inhibited granulocyte-macrophagecolony stimulating factor-included proliferation of TF-1 cells.

D. Expression of Feline IFN-alpha in E. coli and Bioactivity Thereof

The nucleic acid molecule encoding the mature feline IFN-alpha proteinhaving SEQ ID NO:171 was ligated into the λcro plasmid vector, usingtechniques as described in Example 6B, to produce recombinant moleculepλcro-nFeIFNa_(498e). The recombinant molecule was transformed into E.coli, using techniques similar to those described in Example 6B toproduce recombinant cell E. coli:pλcro-nFeIFNa_(498e). The recombinantcell was grown and induced as described in Example 6B. The resultingfeline IFN-alpha protein, E. coli-expressed PFeIFNa_(166e), which wasexpressed as an insoluble form, was solubilzed using urea and DTT andrefolded using techniques known to those skilled in the art. Therefolded E. coli-expressed feline IFN-alpha protein PFeIFNa_(166e) whentested for antiviral activity as described in Example 7C was found tohave significant antiviral activity.

Example 8

This example describes the isolation and sequencing of felinegranulocyte-macrophage colony-stimulating factor (GMCSF) nucleic acidmolecules and proteins of the present invention. This example alsodescribes expression of a feline GMCSF protein of the present invention.

Nucleic acid molecules encoding feline GMCSF were isolated as follows. AcDNA library was prepared from feline PBMCs stimulated with Con A for 12hours, as previously described in Example 2. An aliquot of this librarywas used as a template to amplify feline GMCSF nucleic aid molecules byPCR using Amplitaq DNA polymerase™ (PE Applied Biosystems Inc, FosterCity, Calif.) and the following primers and conditions. The sequence ofthe forward primer was 5′ CAGGGATCCA CCATGTGGCT GCAGAACCTG CTTTTCC 3′(SEQ ID NO:149), and that of the reverse primer was 5′ TTACTTCTGGTCTGGTCCCC AGCAGTCAAA GGGGTTGTTA AACAGAAAAT 3′ (SEQ ID NO:150). Thefollowing PCR protocol was used: one initial denaturation step at 95° C.for 5 minutes; then 35 cycles of the following: 94° C. for 30 seconds,then 50° C. for 30 seconds, then 72° C. for 90 seconds; followed by afinal extension at 72° C. for 7 minutes. PCR products were cloned intothe CMV-Intron A vector and the clones were sequenced as described inExample 7.

A PCR product was isolated, referred to herein as nFeGMCSF₄₄₄, thecoding strand of which is represented herein as SEQ ID NO:119, and itscomplement is denoted SEQ ID NO:121. Translation of the open readingframe in SEQ ID NO:119 suggests that nucleic acid molecule nFeGMCSF₄₄₄encodes a protein containing 144 amino acids, referred to herein asPFeGMCSF₁₄₄, with an amino acid sequence denoted SEQ ID NO:120, assumingan open reading frame in which the first codon spans from nucleotide 10through nucleotide 12 of SEQ ID NO:119, and the stop codon spans fromnucleotide 442 through nucleotide 444 of SEQ ID NO:121. The encodedprotein has a predicted molecular weight of 16 kDa. The coding regionencoding PFeGMCSF₁₄₄ is presented herein as nFeGMCSF₄₃₂ which has thenucleotide sequence SEQ ID NO:122 (the coding strand) and SEQ ID NO:123(the complementary strand). A putative signal peptide cleavage site isbetween amino acid positions 17 and 18, based on homology with human,mouse and bovine GMCSF proteins. The nucleic acid molecule encoding theproposed mature protein is denoted as nFeGMCSF₃₈₁ and has a nucleotidesequence represented herein as SEQ ID NO:124 and a complementarysequence represented herein as SEQ ID NO:126. The amino acid sequence ofthe putative mature protein, referred to herein as PFeGMCSF₁₂₇ has anamino acid sequence represented herein as SEQ ID NO:125. The number ofamino acids in the feline GMCSF protein is the same compared to human,porcine, ovine and canine GMCSF proteins. The feline GMCSF protein isone amino acid longer than bovine GMCSF and three amino acids longerthan murine GMCSF.

The deduced amino acid sequence of the full-length feline GMCSF proteinof the present invention has four non-conservative changes and oneconservative change compared to a GenBank entry for feline GMCSF(accession #AF053007). Amino acids asparagine, methionine, threonine,and lysine at positions 10, 36, 56 and 126 of the GenBank entry havebeen changed to glycine, isoluecine, alanine and asparagine,respectively, in PfeGMCSF₁₄₄, PFeGMCSF₁₄₄, containing the above-notedamino acid substitutions, appears to have GMCSF activity, asdemonstrated by an experiment in which supernatant collected fromChinese Hamster Ovary (CHO) cells that were transiently transfected witha recombinant molecule encoding a feline GMCSF protein of the presentinvention was able to include proliferation of TF-1 cells.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. An isolated protein selected from the group consisting of: (a) anisolated protein of at least about 20 amino acids in length, whereinsaid 20 amino acids are encoded by a nucleic acid molecule that has anat least 60 contiguous nucleotide region identical in sequence to a 60contiguous nucleotide region of a nucleic acid sequence selected fromthe group consisting of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ IDNO:91, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, and SEQID NO:104; and (b) an isolated protein of at least about 20 amino acidsin length, wherein said protein has an at least 20 contiguous amino acidregion identical in sequence to a 20 contiguous amino acid regionselected from the group consisting of SEQ ID NO:92, SEQ ID NO:97, SEQ IDNO:100, and SEQ ID NO:105, wherein said isolated protein of (a) or (b)elicits an immune response against a canine IL-13 protein or has IL-13activity.
 2. The isolated protein of claim 1, wherein said protein hasan amino acid sequence selected from the group consisting of SEQ IDNO:92, SEQ ID NO:97, SEQ ID NO:100, and SEQ ID NO:105.
 3. The isolatedprotein of claim 2, wherein the protein has the amino acid sequence ofSEQ ID NO:92.
 4. The isolated protein of claim 2, wherein the proteinhas the amino acid sequence of SEQ ID NO:97.
 5. The isolated protein ofclaim 2, wherein the protein has the amino acid sequence of SEQ IDNO:100.
 6. The isolated protein of claim 2, wherein the protein has theamino acid sequence of SEQ ID NO:105.
 7. An isolated protein having anamino acid sequence that is at least about 85 percent identical to anamino acid sequence selected from the group consisting of SEQ ID NO:92,SEQ ID NO:97, SEQ ID NO:100, and SEQ ID NO:105, wherein said isolatedprotein elicits an immune response against a canine IL-13 protein or hasIL-13 activity.
 8. A therapeutic composition comprising the isolatedprotein of claim
 1. 9. The composition of claim 8, wherein saidcomposition further comprises a component selected from the groupconsisting of an excipient, an adjuvant and a carrier.
 10. A method toregulate an immune response in an animal comprising administering to theanimal the therapeutic composition of claim
 8. 11. The method of claim10, wherein said animal is a canid.
 12. The method of claim 10, whereinsaid composition further comprises a component selected from the groupconsisting of an excipient, an adjuvant and a carrier.